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1. ii Integrated Excision Tool x p rMember image E xK 1 401 201 Demo 30degree2 141045 1 1 24 Jan 20 Completed _ i 1 402 2403 Demo 30Odegree2 14153 0 1 1 24 Jan 20 Completed im 1 4 1302 Demo 30degree2 198995 1 1 24 Jan 20 Completed i 1 404 6102 Demo 30degree2 23351 1 1 1 K 1 Al 4301 emo 30degree2 247859 1 1 2 1 406 2503 Demo 30deqree2 469602 1 5 gt Cutter image i 1 407 3502 Demo 30degree2 576730 1 Ei a ES ES e na na na sectation a r it rSetup A oo RRRA ala Simulate X Cutter X Camera Fig 9 15 Cutting progress As each cut is being made it will be highlighted in the table list In progress or Completed displays in the Status column The checkbox in the Cut column next to that spot will be automatically deselected The spot being cut displays in the Member Image window and in the Cutter image window The spot in the Cutter image window is filled with yellow while it is being cut and surrounded by a green circle when the cut is complete This process takes a while so please be patient 9 29 PDQuest User Guide 1 Integrated Excision Tool UN xj Cut list Analysis set name Demo Qual Gel spots to cut 1 0 Analysis set spot count f 2 Total cuts incl multi 1 0 Cut MTP SSP Gel Quantity Run Cuts Cut date Status Lj 1 401 4202 Demo 30degree 3204 5 1 05 Dec 2002 Completed 2 Lj
2. Close door and click Acquire Image Acquire image tenet Step 2 Examine Image ee ale Mes Lm can see a visible gel plug qe qe qt a Step 3 Select Action OA A Click on Specify re cuts below to repeat any cuts a t The image of the microtiter plate will remain on the screen BE OOOO CC during re cut selection 2 101002039 AAAI of Specify re cuts X Nore cuts S l Fig 9 20 Microtiter plate after zoom and transform functions applied Step 8 If any of the wells are empty and you want to re cut those spots click on Specify re cuts Then follow these on screen instructions 1 POQUEST Ea A Remove Microtiter Plate Return the microtiter plate to the microtiter plate holder Remove Gel Cutting Sheet from the cutting stage Be careful to not move the gell OF Fig 9 21 Message window before recuts are started Step 9 Return the microtiter plate to the plate holder 9 34 Integrated Excision Tool Step 10 Remove the plastic sheet from the top of the gel Step 11 Do not move the gel The image and spot selections that were already made still apply to the gel as long as it does not move Step 12 After removing the microtiter plate click OK on the message The Image Microtiter Plate window will close and an image of the microtiter plate will open in front of the Basic Excision Tool screen If you need to resize the window close the recut
3. Show description Relate Column to Category Colimn 1 SSF Column color Column 3 size of Import ES Cancel Load Settings Save Settings E Help Fig 7 13 Import Annotations Tool Select a file to import by clicking the folder icon to the right of the field The next two fields are informational only If you have the First line is Header checked then the Header row appears in the Header line field The first data line shows your first line of data 2nd line of text in your file if you have first line is header selected If your text file is tab delimited columns separated by tabs the tabs appear as boxes between the data Confirm Overwrites If any annotations will change when you click import you will get a warning message asking you to confirm the change 7 29 PDQuest User Guide i POQUEST X AN confirm Overwrite Would you like to overwrite the existing annotation Spot 1502 was clear now blue wf Yes 2 No Fig 7 14 Confirm Overwrite Dialog If you do not want to overwrite what is there already click no and the import will skip that annotation and continue If you click yes any data will be overwritten If the Confirm overwrites box is unchecked changes are made to existing information without a warning Column delimiter This option determines how columns are separated in your text file Currently your only options are comma and tab Your text fil
4. Gel Name PAIN Analysis Set Qty Valid Spot Oty Total Density Specified Value Counts Loaded Comp femmes asoman o O O E Pfft 00 J Done X Cancel Help Fig 6 19 Normalize dialog box for member images In the dialog box check the Enable Normalization box to activate normalization for the MatchSet Normalization Formula Normalization in PDQuest is performed for each gel in a MatchSet using the following formula Normalized spot quantity Raw spot quantity Scaling factor Pipetting error compensation factor if any Normalization factor Raw spot quantity is the unnormalized quantity intensity of each spot Scaling factor is a factor you select to give a meaningful value see below and Normalization factor is calculated for each gel based on the normalization method you select see below 6 24 Analysis Tools Pipetting error compensation is only available if you have calibrated the gels using Calstrips and entered the number of counts loaded in the gel into the Gel Record See Appendix J After you have selected the settings described below click Done 6 7 a Scaling Factor The scaling factor is a constant by which the normalized quantity is multiplied to give a more meaningful value A common scaling factor is 10 giving units of parts per million PPM Under Scaling specify the scaling factor that you would like to use by clicking on PPM x 1000000 Percent x 100 or User Spe
5. Fig 5 3 Create MatchSet dialog At the top of the Create MatchSet dialog enter a name for the MatchSet in the Name field To assign your MatchSet a default name the default prefix can be set under Edit gt Preferences click the Auto button Enter a description of the MatchSet in the text field next to the Description prompt You can enter further information about the MatchSet in the comments field Next specify whether you are creating a Level 1 MatchSet or a Higher Level MatchSet Level 1 MatchSets are created from the Gaussian images of scanned gels Higher Level MatchSets are created from other MatchSets see section 5 8 b Higher Level MatchSets Matchsets If you are creating a level 1 MatchSet use the Add button to select the Gaussian images to be included in the MatchSet The names of the files you selected will appear in the Members list Click the Add button to open the Select New Member dialog where you can navigate to the folder in which your gel images are stored You can select more than one new member by holding down the Shift key and clicking on the images you want to include Select New Member sample ve x1 gsc S sample v2 gim sample v2 gsp samples v1 x1 gsc A samples 1 gir samples v1 gsp E sampled v1 x1 gsc 5 samples v1 x1 gsc E sampled v1 x1 gsc File name Files of type all files Cancel ve Fig 5 4 Select New Member dialog The Select New Mem
6. 200 micrometer 800 micrometer i Image file size 13 73 Mb K E QOreaie GB seo A Options N uto Save After Scan _ Highlight Saturated Pixels OE E Hide Grid P x warm On Saturated Pixels Q Info Help ba S E U 10 11 12 13 14 15 16 Fig l 2 Personal FX acquisition window The default scanning window is marked by grid lines that divide the area into quadrants There is also an outer box and inner box marked by thicker lines The quadrants are numbered 1 16 left to right and lettered A U top to bottom If you prefer a scanning window measured in centimeters deselect the Quadrant Mode checkbox in the control panel by clicking on it To hide the gridlines click on the Hide Grid checkbox under Options The control panel has been arranged from top to bottom to guide you through the acquisition procedure There are three basic steps to scanning an image using the Personal FX 1 Select the scan area PDQuest User Guide 2 Select the resolution 3 Acquire the image 2 Step l Select Scan Area To select a scan area drag your mouse within the scanning window In the scanning window your cursor appearance will change to a cross The border of the scan area you are selecting is marked by a frame Fig I 3 Selecting a scan area If you are in quadrant mode note that the frame locks onto the next quadrant as you drag When you releas
7. Fig N 7 Open From WorksBase Once you select a record at a given level it filters the records below it This is indicated by the appearance of the filter icon to the left of the level selection button PDQuest User Guide For example if you want to see all the experiments in a certain project you would first select the project then press the Experiments level button The list will show only the experiments in the selected project If at that point you want to see all experiments not only those in the selected project press the filter button next to the Projects level button It will become a down arrow indicating pass through without filtering The main database window in the Open from WorksBase dialog box changes depending on your menu selection For example if you clicked the Samples button the window displays the Sample Name Sample Acquisition Date and the Sample Volume Open images or MatchSets by clicking the Open Images or Open MatchSets buttons To view arecord s information click View To edit the record click Edit At any level once you have selected one or more records press Open Images s or Open MatchSet s to open the images or MatchSets associated with the selected record s For example if you want to open all the images in a given project you would 1 Press the project level button all the projects in the database are listed 2 Select the project of interest 3 Press the Open Image
8. O Ok Cancel Fig G 9 Options dialog box G 6 a Dark Subtraction Type All CCD cameras accumulate electrons that produce a signal that is indistinguishable from light This dark count adds to the noise in your images In most cases you will want to subtract this dark count from your images Normal The Normal option button selects the default dark subtraction type In this mode after you acquire an image a dark image of the same exposure length will be taken and this will be subtracted from your image The progress of the dark exposure will be displayed in the Exposure Status bar following the regular image exposure Note In Normal mode a dark image is only acquired the first time you perform a scan with particular application and exposure settings If you perform subsequent scans with the same settings no dark exposure will be taken G 12 Appendix G Fluor S Referenced If you do not want to perform a dark exposure with each acquisition you can take a reference dark exposure that will be saved and subtracted from all subsequent acquisitions Click on the Referenced button to activate this feature The first time you acquire an image after selecting this option the Fluor S will take a 60 second dark exposure that will be saved and used to subtract the dark count from all subsequent acquisitions For image exposures of greater or less than 60 seconds the reference dark will be scaled acc
9. asc Background color arial a gt Black gt money OF Cancel Help OK _ Cancel _ Hep Fig 3 25 Text Overlay Properties dialog box To enter your text simply begin typing in the main field The buttons in the dialog allow you to select the properties of your text including format alignment and justification The pull down boxes from left to right allow you to select the font style font size color of the text and color of the background within the text box Once you have typed your text click OK When you exit the Text Overlay Properties dialog by clicking OK the text you typed will appear on the image at the spot where you originally clicked You can create as many textual overlays as you want Editing a Text Overlay To edit a text overlay make sure the Text Tool or Select Tool is assigned to your mouse then double click the overlay to open the Properties dialog The existing text will be displayed and can be edited 3 39 PDQuest User Guide Line Tool You can use the Line Tool on the Text Overlay toolbar to draw a line between text and an image feature or between any two points of interest on your image Click the Line Tool button then drag on your image to create the line You can create as many lines as you want To resize or adjust a line make sure the Line Tool or Select Tool is assigned to your mouse then position your cursor on one end of the line marked by a circle and dra
10. y Ae Experiments al Eje Samples 2 2nd Dim Gels gt Lar Programs Unfiltered 4 Projects Unfiltered Unfiltered Unfiltered Unfiltered Sua ETE Matchsets Unfiltered a Sample Name Sample Acquisition Date Sample Volume 30d e 30d e 30d e 30d e 30d e 30d e 30d e 30d e 30d e 30d e 30d e 30d e 30d e 30d e 30d e 30d e 30d e 30d e 30de 30d e coli Fraction 10 coli Fraction 10 coli Fraction 11 coli Fraction 11 coli Fraction 12 coli Fraction 12 coli Fraction 13 coli Fraction 13 coli Fraction 14 coli Fraction 14 coli Fraction 2 coli Fraction 2 coli Fraction 3 coli Fraction 3 coli Fraction 4 coli Fraction 4 coli Fraction 5 coli Fraction 5 coli Fraction 6 coli Fraction 6 22 Jan 2003 22 Jan 2003 22 Jan 2003 22 Jan 2003 22 Jan 2003 22 Jan 2003 22 Jan 2003 22 Jan 2003 22 Jan 2003 22 Jan 2003 22 Jan 2003 22 Jan 2003 22 Jan 2003 22 Jan 2003 22 Jan 2003 22 Jan 2003 22 Jan 2003 22 Jan 2003 22 Jan 2003 22 Jan 2003 0 000000 0 000000 0 000000 0 000000 0 000000 0 000000 0 000000 0 000000 0 000000 0 000000 0 000000 0 000000 0 000000 0 000000 0 000000 0 000000 0 000000 0 000000 0 000000 0 000000 Open Image s EJOpen Matchset s Cancel 4 Help E ua jf gt Records in page 500 selected o SY Filter
11. 1 2 Introduction Signal intensity of a single pixel intensity M FT if ji r Fig 1 1 Representation of the pixels in two digitally imaged spots in a gel For a data object to be visible and quantifiable the intensity of its clustered pixels must be higher than the intensity of the pixels that make up the background of the image The total intensity of a data object is the sum of the intensities of all the pixels that make up the object The mean intensity of a data object is the total intensity divided by the number of pixels in the object The units of signal intensity are Optical Density O D in the case of the GS 700 and GS 710 densitometers the Gel Doc and ChemiDoc with a white light source or the Fluor S and Fluor S MAX Multilmagers with white light illumination Signal intensity is expressed in counts when using the Personal FX or FX or in the case of the Gel Doc ChemiDoc Fluor S or Fluor S MAX when using the UV light source PDQuest User Guide 13 PDQuest Workflow Acquire the image Size and orient the image Spot identification Spot comparison and matching y Data analysis Spot cutting and mass spec analysis Publish results Fig 1 2 Steps involved in using PDQuest Image Acquisition PDQuest can acquire images of gels using Bio Rad s densitometers storage phosphor imagers and camera based imaging systems First you open the acquisition window for your imaging dev
12. Description Directory C Program Files Bio Rad he Discovery Series oample Images Dii Filename samples ve x1 qsc Scan Date 21 Jun 1999 14 21 scanner Melanie File Imported Pixel sizefumj 175 0 Y 175 0 scan Areatmm 491 3 Y 145 1 Data Range 4095 00 Int scan Pixels A S22 Y 029 Memory size 974 635 kb scan History 29 Jul 1999 08 56 Horizontal Flip 29 Jul 1999 10 27 Horizontal Flip alel g tax e Page 1 of 2 gt e Fig 2 15 Master Image Report The report includes the following information 2 23 PDQuest User Guide The image title A description of the image if you specified one The directory location The filename of the image The type of imaging device scanner Pixel size by X and Y axis as measured in um The scan image area in terms of X and Y axis and the data range as measured in integers The number of pixels scanned and the memory size as measured in Kilobytes The Scan history date and time of the report and the action s performed during that scan The imaging area and number of pixels The intensity range image color and memory size Image background information Relevant lane and band information To print a image report of a particular gel select Image Report from the File gt Print submenu This opens the standard print dialog box see Print Image above Note 2 5 0 TIFF images do not contain all the tagged information that would normally be included in an i
13. Transform Linear 8 bit Entire image 16 bit e Current view 24 bit AGE f Current view Size Pixels 253 Y 410 Image 103 70 Eb Export X Cancel H Help Fig 2 10 Export to TIFF dialog Note See Filtered Images and Gaussian Images for a description of the different image types In the export dialog first select whether you want to export the raw data image Analysis option or the currently displayed view of the image Publishing option Select the appropriate radio button to publish the export view excluding or including overlays Analysis Export 2 14 General Operation The Analysis option exports the image data unmodified by any viewing adjustments you may have made such as Transform or Zoom If you select this option the other controls in the dialog box will become inactive Publishing Export If you select the Publishing export option the exported TIFF image will look like the image as it is currently displayed on the computer screen You can specify a resolution for the exported image by selecting 72 dpi typical computer screen resolution 150 dpi or 300 dpi standard printing resolutions Same as scan or any resolution you specify up to the resolution of the scan If you have log transformed the image you can specify a Linear transform or preserve the Current view If your image has a pixel depth of 16 bits you can compress it to 8 bit data for export to TIFF b
14. onto the next quadrant as you drag When you release the mouse button the border changes to a dashed blue line indicating a selected area To reposition the scanning box you have selected position your cursor inside the box and drag The entire box will move To resize the box position your cursor on a box side and drag The side you have selected will move To redo the box entirely position your cursor outside the box and drag The old box will disappear and a new box will be created J 11 PDQuest User Guide You can also select the scanning area by entering coordinates in the appropriate fields Top Bottom Left Right After you enter a coordinate the position of the scanning area box will change accordingly When selecting be sure to include the entire area of interest and be generous with borders You can always crop the image later J 4 Step Ill Select Resolution The FX acquisition window allows you to scan at 50 100 200 or 800 micrometers These resolutions are listed as option buttons in the control panel Step Ul Select Resolution 50 micrometer f 100 micrometer gt 200 micrometer G00 micrometer Image file size 3 19 Mb Fig J 10 Resolution option buttons The resolution you select should be based on the size of the objects e g bands spots you are interested in For example 50 micrometer resolution should be reserved for images requiring the highest level of detail e g high
15. 0 21 0 30 13 1 91 2 70 0 37 Oo 52 14 2 08 2 51 0 53 0 75 15 2 21 3 13 3 4 Be 0 68 0 96 16 2 38 3 34 E 0 83 1 17 7 z sz 3 56 5 3 0 993 1 40 18 2 68 3 73 1 14 1 61 BY ERE 4 00 1 23 1 82 0 2 98 4 21 10 1 45 2 05 2 3 13 4 43 11 1 80 2 26 OF Cancel H Help Fig F 10 Step Tablet dialog box Attached to the outside of your GS 800 you will find a copy of the manufacturer s printout of the diffuse density values for each segment of the transmissive step tablet as well as a serial number for the tablet These exact density values must be entered into the software to associate a correct density value with each step on the step tablet Note Scanning in transmissive mode with incorrect step tablet values can cause significant errors in the reported densities of your scans First type the serial number for the tablet into the Tablet Serial Number field The Quantity Offset field does not apply in the GS 800 in transmissive mode This value should remain at zero Next enter the step tablet values into the appropriate fields under the Diffuse column After the step tablet is scanned the software will associate each density value with its corresponding segment on the step tablet The density values do not need to be reentered each time you calibrate When you are finished entering the transmissive step tablet values click on OK F 11 PDQuest User Guide Reflective Step Tablet
16. 3 3 Assigning and Interchanging Images You can use the Assign and Interchange commands to select the image you want to display in a particular window or subwindow Assigning Images A To assign an image to a particular subwindow select the Assign tool o from the main toolbar or Window menu and click in the subwindow or use the Ctrl A key command in desired subwindow This will open a pop up list of all the images currently loaded in the MatchSet or scanset Select an image name from the list the image will appear in the selected window samplel wl Filtered samplel wl Gaussian samplel wl ul Raw D Image samplel yl Filtered i reps Se pr V1 Gaussian Fig 3 2 Assign Image lists all the loaded images select one to display it Interchanging Images Interchange Images switches between the Raw 2 D scan Filtered image and Gaussian image of the same gel within a selected subwindow With the images loaded select the tool from the main toolbar or View menu and click in the subwindow or use the F11 key command in the subwindow The type of image currently displayed is listed at the bottom of each subwindow Interchange All Images Shift F11 View menu switches between the Filtered images and Gaussian images in all the displayed subwindows of a MatchSet 3 3 PDQuest User Guide 3 4 Tiling Windows The tile commands under the Window menu are used to arrange image windows on the screen Note The tile command
17. AAA ul g 3 ee Crop 4 mm E da aoe D xe Si E y Advanced Crop Define Crop rea Define Calstrip Area Vertical Flip Place Erosshair Horizontal Flip Load Crop Settings Rotate Save Grop Settings Filter Wizard Delete Crop Settings Filter List Invert Data Reduce File Size Fig 3 17 Crop commands 3 26 Viewing and Editing Images Select Crop from the Image menu or toolbar and drag the cursor on the image creating a box Everything outside the crop box will be deleted The size of the crop area in millimeters and number of pixels is shown at the bottom of the crop box as is the file size of the image inside the crop area e To reposition the crop box position your cursor at the center of the box The cursor appearance will change to a multidirectional arrow symbol You can then drag the box to a new position e To resize the box position your cursor on a box border or corner The cursor appearance will change to a bidirectional arrow You can then drag that border or corner in or out resizing the box e To redraw the box position your cursor outside the box The cursor appearance will change back to the Crop tool and you can draw another box replacing the one you just drew To complete the crop position your cursor inside the box slightly off center The cursor appearance will change to a scissors symbol Click once to perform the crop A pop up box will ask you whet
18. At the beginning of each row is a Cut checkbox specifying the spots that will be cut in the current cut run Uncheck the box if you do not want to perform that cut After each cut is performed its status will be listed as completed and its cut checkbox will be deselected After a cut run is complete you can reselect checkboxes in the list to recut any missed spots or cut spots that were skipped in the previous run Step 2 Select the Cut Run Options Before beginning a cut run using the Integrated Excision Tool you must select the appropriate cutter options for that run Select Spin When Cutting to make the cutting tip spin as it cuts This 1s recommended for all cutting including gels and blots Deselect this option if you want to test X Y positioning without cutting Spin When Ejecting will cause the cutting tip to spin as it ejects the cut into a microplate well This is only recommended for PVDF or nitrocellulose blots Cut Longer will cause the cutting tip to hold in the down position longer when making a cut Double Eject will cause the eject pin in the cutting tip to eject twice into a microtiter plate well This is useful for membrane blots if the membrane seems to be sticking to the cutting tip No spin when lifting stops the tip from spinning after the cut is made and the tip is lifted from the gel This option may be useful for some gel or blot applications 9 19 PDQuest User Guide Check the Backed gel se
19. Data Handling The Densitometer will be told to scan at its maximum resolution and spatial averaging will be used to produce the desired resolution This gives better spatial fidelity and lower noise although it takes longer to scan JOverzample A modified logarithmic scaling system will be used to reduce Save Scaled Data the size of the data by 50 It introduces a constant relative error of less than 3 Cancel Help Fig D 9 More Options in the GS 700 acquisition window To enable automatic calibration click on the Calibration On checkbox Note Before you can calibrate you must enter the correct step tablet values for your transmissive calibration strip into the Step Tablet Values form as described below You do not need to change the values in the reflective step tablet form you can use the default values With Calibration On selected the other calibration settings become active D 10 Appendix D GS 700 Calibration Strip Window When calibration is turned on a calibration strip window will appear below the main scanning window and the length of the main scanning window will be reduced to 29 cm reflective and 23 cm transmissive Every time the densitometer calibrates an image of the calibration strip will appear in the calibration strip window D 6 a Editing the Step Tablet Before you can calibrate you must enter the step tablet values for your transmissive calibration strip into the Step Ta
20. Hee r E Y OK apoyan Auton Cancel C Hep Awoscae Feset image cotor Frequency Distribution histogram Fig 3 15 Transform dialog box You can use regular viewing tools like Zoom Box and View Entire Image in the preview window to focus on particular regions of interest You can also use Grab tool and arrow keys to move the image within the preview window The Frequency Distribution histogram shows the total data range in the image and the amount of data at each point in the range In a typical scan there is a signal spike at the left gray end of the histogram due to background noise The Transform Plot is a logarithmic representation of how the raw pixel data are mapped to the pixels of your computer screen 3 22 Viewing and Editing Images Note If you are using the Transform window in conjunction with the Multi channel Viewer you can adjust the display of each channel independently by selecting the appropriate channel option button below the preview window 3 12 a Transform Controls Auto scale Click the Auto scale button to optimize the selected image automatically The lightest part of the image is set to the minimum intensity white and the darkest is set to the maximum intensity black This maximizes the display range for the image You can then fine tune the display using the sliders Select the Always Auto scale checkbox to automatically auto scale every image that you open This setting
21. Loh Log Transform window Gb _ X n_ _lhl EZ erase y r fipa GG eee jea 1940 Mao tates p revert ieai fj Log High Low Skiers fn Hri fiosa DO Hioh ght saturated piali Danma qq A p Y 0 Coca Dv ceo Mo SERTI Y xi leg Joa Cai E aira a a fe pagr E D aih hs gaai Lap 1 4 Cn Dip Tel hrs mb rg Cs bng Wh ts ing l Db Di aer bar MESTRE ia as Ag Fey d Fe le bs Epi Dime Fim Hie heed rr loz METAS Tal ae Car mr pa ll Ler Tz fetes i Coomas color selected alla Main window in Basic excision tool is transformed after spots F have been selected for cutting i m Q ts Fig 8 6 Transform window with Coomas color selected The colored box displayed on the image shows the boundary of the cutting area The cutting head cannot cut spots outside this area To select another spot to cut position your cursor in the center of the colored box and drag it over to the new spot 8 11 PDQuest User Guide Spots selected outside the cutting area cannot be cut Either remove the cut or locate it inside the cutting area 8 2 0 Step 2 Specifying Cuts In this step you click on spots in the image to identify which spots to cut Use the Zoom Box tool on the right of the Basic Excision Tool to magnify each spot When clicking on spots in the image select the Snap to Peak checkbox to automatically center the cut circle on the spot peak calc
22. Mark spots on e Master gel Method f Ho spots o Add spots ta set All spots in member Se Remove spots from set Operand gels All gels Saturated spots Show x Window labels J S5P number Import list of SSP from text file Format Spot count o wf Save X Cancel 7 Help Fig 7 6 Arbitrary Analysis set No Spots The No spots option creates an empty analysis set to which you can add any spots you choose To manually add spots to the analysis set Click the Add spots to set button Click on a spot to add it to the set The spot will be highlighted indicating that it has been added to the analysis set To remove a spot click the button labeled Remove spots from set then click a spot in the image to remove the spot 7 16 Analysis Sets and Annotations All Spots in Member The All spots in member option will create an analysis set containing all the spots ina selected member that are matched to the Master Click the drop down button to the right of the member field to display a list of the members from which to choose The member you chose displays in the Member field The analysis set will include all the spots in the member you selected that are matched to the Master Saturated Spots Saturated spots will create an analysis set of all spots that are saturated in any member Importing Spots from External Analysis PDQuest allows you import a set of spots from another Mat
23. PDQuest User Guide for Version 7 1 1 Windows and Macintosh P N 4000129 15 RevA PDQuest User Guide Bio Rad Technical Service Department Phone 800 424 6723 510 741 2612 Fax 510 741 5802 E mail LSG TechServ US O Bio Rad com Notice No part of this publication may be reproduced or transmitted in any form or by any means electronic or mechanical including photocopy recording or any information storage or retrieval system without permission in writing from Bio Rad PDQuest and The Discovery Series are trademarks of Bio Rad Laboratories All other trademarks and registered trademarks are of their respective companies WASTE text engine 1993 1997 Marco Piovanelli Limitations of Liability Bio Rad is not responsible for the misinterpretation of results obtained by following the instructions in this guide Whenever possible you should contact the Technical Services Department at Bio Rad to discuss your results As with all scientific studies we recommend that you repeat your experiment at least once before making any significant conclusions for presentation or publication Copyright 2003 by Bio Rad Laboratories All rights reserved Table of Contents 1 INTOdUCUON sa 1 1 A AOVETVIE WOE PDOUCSE so TSE 1 1 132 Digital Data and Signal Intensa dt 1 2 1 3 PDQuest Workflow anti di ia 1 4 LA Computer Require Ments lt 2 255 hassscecnantar ne a onde masareaetanetees 1 7 LSe ni e ROTA 5 Steed e E E un A 1 9 15
24. Simulate X Cutter 4 Camera x No spin when lifting JBacked gel Tip motion range E o 3 Gel defaults PVDF defaults Select microtiter plate Begin Resume lt 5 Pause E Confirm g4 Align Export Close Help Fig 9 7 Integrated excision tool 1 Select the Cut List In this step you select the cut list created using the Excision Gel Selection tool and specify which cuts you want to perform for this run 2 Select Cutter Options In this step you select the options for the current cut run 3 Select Microtiter Plate In this step you select a microtiter plate and specify the starting well 4 Align the Images Next you click the Align button and align the first MatchSet image and the spot cutter image 5 Finally you perform the cut run When the run is complete you can recut spots if necessary 9 18 Integrated Excision Tool Step 1 Select the Cut List At the top of the Integrated Excision Tool dialog click the pull down button to select from the available cut lists you created using the Excision Gel Selection tool The cut list will be displayed in table format in the middle of the dialog box The cut list displays the microplate well spot number gel spot quantity in that gel cut run number and number of cuts for each spot The cuts are listed in the table in order of gel so that all the cuts will be performed on one gel before you are prompted to load the next one
25. Spots defined using the boundary tools are not Gaussian modeled even though they appear in the Gaussian image However their quantities can be compared with regular Gaussian modeled spots Note Creating a spot boundary will overwrite the results of spot detection and Gaussian modeling However regular spot detection will not overwrite any spots defined using the boundary tools ooo You can open the Spot Boundary Tools toolbar from the main menu or Grea select commands from the Spots gt Spot Boundary Tools submenu Spot Boundary Tools x k Berl pAg Spot Boundary Tools Select Tool Show Crosshairs F5 Spot Contour Tool Show All Crosshairs Shitt F5 Spot Free hand Tool Show Elipeez Al F5 Copy To Clipboard Show All Elli Ctro OW ass dl Paste From Clipboard Fig 4 12 Spot boundary tools Spot Contour Tool To use this tool first magnify the spot you want to quantify in the Filtered image then select Spot Contour Tool from the submenu or toolbar Click and drag the cursor from the inside of the spot outward to change the shape of the contour as you move over pixels of different intensity note that the contour is created by enclosing all pixels whose intensity is greater than or equal to the intensity of the pixel at the cursor When you release the mouse button the boundary is created The contour should completely surround the spot you want to quantify 4 19 PDQuest User Guide 3 19 Select the con
26. With this checkbox selected when you save a scan a backup copy will be placed in the same directory as the scanned image Windows backup files will have an sbk extension Macintosh backup files will have the word backup after the file name Note To open a Windows backup file you must first change the file extension To locate the file after changing the extension select All files in the Files of type drop down list of the Open dialog Highlight Saturated Pixels When this box is checked any saturated pixels in the image will appear highlighted in red in the scan window and in the pop up image window To view hide saturated pixels in the pop up image window use the Image gt Transform command J 14 Appendix J FX Hide Grid To hide the gridlines in the scanning area window click on the Hide Grid checkbox Warn on Saturated Pixels The Warn on Saturated Pixels option is checked by default Uncheck this box to disable this warning PDQuest User Guide Appendix K VersaDoc Fig K 1 VersaDoc Before you can acquire images using the VersaDoc Imaging System you need to install the Roper Scientific interface adapter and its associated device drivers in your computer The driver installation procedures for Windows operating systems inlcude pre installation and use of the Windows Hardware Wizards Note Important Please read and follow the driver installation procedures contained in the README documents befo
27. x Show grdlines yf Done X Cancel Z Help Fig 11 9 Quantity Table Report dialog box You can type a report title the name of the researcher and the name of the research institution into the appropriate fields 11 9 PDQuest User Guide Specify the spots to include in your report All Matched Spots or the spots in a Specified Analysis Set dropdown list If you click Analysis Set click the drop down arrow to display a list of available Analysis Sets The name of the set you choose appears in the field If you want to see spot quantitation for each individual member of the MatchSet select Individual Gel in the Quantitation section If you want to see the average quantitation for a Replicate Group click the Replicate Groups You can sort your spots by SSP number DSN trend ratio molecular weight or isoelectric point by clicking on the appropriate Sort by button To sort in ascending order check the box labeled Increasing Options The following options can be included in the Quantity Table Report e If DSN numbers are assigned to the protein spots and you would like to see them on the report click the DSN button e MrpI data will be reported if you click the MrpI button e Check the box labeled Trend to view the trend of each spot e You can view the group count and the group critical value by checking Group Count and GroupCV repectively e You can view the ratio of spots to a specific gel by clicking Ratio
28. 1 402 1303 Demo 30deqree 3509 5 1 05 Dec 2002 Completed x 1 403 6201 Demo 30degree2 2567 1 1 x 1 404 5401 Demo 30degree2 4185 7 1 x 1 405 9701 Demo 30degree2 6490 9 1 1 1 1 1 1 Member image x 1 406 0602 Demo 30degree2 13572 7 x 1 407 0703 Demo 30degree3 2068 6 x 1 408 4401 Demo 30degree3 2491 9 x 1 409 3704 Demo 30degree3 2560 3 x 1 410 7903 Demo 30degree3 2649 3 Status Cutter options e A M b ob d d obs Misc Options X Multiple cuts spot Wash options Station 1 X Station 2 X Station 3 x Station 4 X Spin when cutting _ Cut longer x Spin when ejecting X Double eject Save validation image Setup asa Simulate X Cutter x Camera CI No spin when lifting Backed gel Tip motion range Gel defaults PYDF defaults Select microtiter plate EU Align O Begin Resume E Confirm EM Close B Help Fig 9 16 Progress completed on six cuts in the cut run Confirming Cuts The Confirm button helps you check the cut progress of a E Sd fluorescent stained gel With visible stained gels it is easier to look in the spot cutter to confirm that the cutting tip is cutting the spot accurately Since the spots are not visible without UV illumination in a fluorescent gel the Confirm function provides an on screen method for monitoring the cut run After one or more cuts have been made clic
29. 6 21 Mrpl commands Note PDQuest needs to know the pH gradient of the gel in order to calculate isoelectric point values To enter the correct orientation go to Edit gt Preferences gt Applications tab and click either Acid Base or Base Acid next to the pH Gradient prompt 6 29 PDQuest User Guide Entering Mrpl Values for Known Spots To determine MrpI values for all the spots in the MatchSet you first need to enter the values for a few known spots Select Enter MrpI Data from the Analysis gt MrpI Standards submenu or toolbar then click on a spot in the MatchSet Master whose molecular weight and or isoelectric point values are known A dialog box prompts you to enter the values for that spot ai POQUEST A Enter Mr and pl fe g 4205 42 lo o o oo Cancel Fig 6 22 Pop up box in which Mrpl data are entered Type the molecular weight first followed by a space followed by the isoelectric point Click OK when you are done If you make a mistake entering the data you can select Enter MrpI Data again and click the spot The values you entered will appear in the dialog box and you can make the necessary corrections If you want to remove the MrpI data for a spot select Remove MrpI Data and click the spot Displaying Mrpl Data To see the proteins for which you have entered MrpI data you can use either the Mark MrpI Spots or Display MrpI Data option The first option highlights the spots for which you
30. Appendix E GS 710 E 3 Step Il Select Scan Area Preview Scan Before selecting the particular area to scan you can preview the entire scanning area to determine the exact position of your sample Click on Preview Scan A quick low resolution scan of the entire scanning area will appear in the scanning window Your sample should be visible within a portion of this scan Selecting an Area Using the preview scan as a guide select your scan area by dragging your mouse within the scanning window The border of the scan area you are selecting will be marked by a frame Note The scan area you select must be at least 1 cm wide When you release the mouse button the border changes to a dashed blue line indicating a selected area e To reposition the scanning area box you have created position your cursor inside the box and drag The entire box will move e To resize the box position your cursor on a box side and drag The side you have selected will move e To redo the box entirely position your cursor outside the box and drag The old box will disappear and a new box will be created You can also select the scanning area by entering coordinates in the appropriate fields Top Bottom Left Right As you enter a coordinate the position of the scanning area box will change accordingly When selecting be sure to include the entire area of interest and be generous with borders You can always crop the image later E 7
31. For the reflective step tablet 1t is recommended that you use the default target values in the software Although you can edit these values based on your own measurements and testing the default values have been preset based on careful testing and review To view or edit the default reflective values make sure that the Reflective checkbox is selected in the main acquisition window then click on the Options button In the Densitometer Options dialog click on the Edit Step Tablet button The Step Tablet Values for GS 800 Reflective dialog box will open When you are finished viewing editing the reflective step tablet values click on OK Diffuse Versus Specular O D In the step tablet form you enter O D as diffuse density and then the software automatically calculates the specular density Specular density is a measure of the light that passes directly through a medium Diffuse density includes light that is scattered as it passes through the medium Step tablet values are given in diffuse density but are measured by the scanner in specular density and therefore must be converted according to the specular diffuse O D ratio This conversion does not affect quantitation q Specular Light passes directly through medium lt q Medium gel film etc r4 NX lt a Diffuse Light is scattered by medium Fig F 11 Specular and diffuse density Diffuse density values are converted to specular optical density units acc
32. General Operation Click the Application tab to access these preferences Enter up to three letters for the MatchSet Name Prefix and a number for the MatchSet Name Sequence When you create a MatchSet you can give it a default name The MatchSet Name Prefix field determines the prefix of the default name while the MatchSet Name Sequence determines the number added to the prefix to complete the name The sequence number increases by each time a new MatchSet is created using the default name Once the prefix and the sequence number have been initially set you probably will not need to change them If Auto match after Landmark is selected PDQuest will auto match all gels in the MatchSet each time you place a landmark in all the gels Finally indicate the orientation in which your gels are run by clicking on either the Acid Base or Base Acid button next to the pH Gradient prompt 2 74 Imagers Checking the name of a scanning device in the Imagers tab allows you to scan using that instrument See the individual appendices on each imaging devices for more details 2 43 PDQuest User Guide fh Preferences l 7 a x Misc Paths Display Toolbars Application Imagerz Please select the Imagers which should be diplayed in the file menu x Gel Doc x Fluor 5 x Personal Fes x Chemi Doc x Fluor S Mae gt Fes x Fluor S Masz x G5 700 x VersaDoc 2 65 210 a 65 800 wf OK X Cancel 7
33. K 10 Appendix K VersaDoc To enable this feature select the Flat Fielding checkbox Note For applications using the UV or white light transillumination you should use the appropriate reference plate to ensure a uniform intensity in the image This will compensate for normal variations in image pixel intensity that occur with a transilluminating light source UV Illumination Flat Fielding When you first select the Flat fielding checkbox and then acquire an image using the UV transilluminator you will be prompted to remove your sample and place the fluorescent reference plate on the VersaDoc sample stage see the VersaDoc User Manual A reference image of the plate will be acquired and saved on your computer hard drive The reference image will be applied to the sample image to generate a Flat Field corrected image White Illumination Flat Fielding When you first select the checkbox and then acquire an image using the white light conversion screen you will be prompted to remove your sample and collect an exposure of the conversion screen A reference image of the screen will be acquired and saved on your computer hard drive The reference image will be applied to the sample image to generate a Flat Field corrected image For subsequent UV or white light trans exposures you will be prompted to either use the appropriate saved Flat Fielding image or acquire a new one Any changes in light source filter or lens setting will require t
34. LSB 2205 Comment PostScript driver Find Printer Fage Range fe All Number of copies fi f Selection Curent Page f Pages Frint Cancel Fig 2 14 Print Image dialog box In the General tab select the printer and page range you want to use In the Layout tab select page orientation duplex printing and advanced options if necessary In the Paper Quality tab set the paper source whether or not you want to print in color and advanced options if necessary When you are satisfied with all the print parameters click Print to send the image to the printer 2 5 b Print Actual Size You can print your images at their actual size using the Print Actual Size command Note If you are using the Gel Doc ChemiDoc Fluor S or Fluor S MAX you must specify the correct image area size when capturing your images to ensure accurate 1 1 printing You can specify the image area size in the acquisition window for the instrument See the chapter on each imaging device for more information 2 22 General Operation Select Print Actual Size from the File gt Print submenu The standard Print Image dialog box will open see previous section 2 5 Image Report Image Report allows you to print out a single page report of an image and its associated information The format of the report is designed to provide a concise yet thorough summary of the most relevant features of an image for documentation purposes
35. PDQuest User Guide E 4 Step Ill Select Resolution To select from a list of possible scanner resolutions click on the Select button under Select Resolution This will open the Select Scan Resolution dialog box Quantity One A f 423 2 212 ala 423 Mb635 635 CAE 423 fe od x od cod 425 105 8 x 105 5 f 105 8 x 423 169 3 x 165 3 MESS x 63 5 Select Scan Resolution Highest For small gels with tiny features Medium high For gels with small features Cancel Asymmetric medium high For gels with tiny bands ele Done High For small gels with ting features Medium For most gels Help Asymmetric medium For most gels with bands Medium low For gels with large features Asymmetic medium low For gels with large bands Low For gels with very large features Asymmetric low For gels with ver wide bards Fig E 7 Select Scan Resolution dialog box Available resolutions are listed from highest to lowest in terms of the dimensions of the resulting pixels in microns Smaller pixels equal higher resolution Each resolution is listed with its typical use In general the size of your pixels should be one tenth the height of your smallest object Some of the resolutions are asymmetrical meaning that the resolution is higher in the vertical dimension 1 e the pixels in the resulting image are larger in the horizontal dimension than in the vertical dimension This is useful for gels with bands w
36. The Open command on the File menu or main toolbar opens previously saved images and MatchSets Note This version of PDQuest will load any images and MatchSets created with earlier versions of PDQuest 2 10 General Operation T x _ _Mso0000 3 2001 09 05 Data samplet 2 gim E ETroll 2001 09 10 13hr 37 min v1 gsc E sample4 2 gsc MS00003 2001 09 05 ms sampled v2 gsp E samplel vl x1 gsc E samples vi x1 gsc sampled vi1 gim samples v1 gim samplel v1 gsp samples 1 gsp samples v2 x1 gsc E sample vi x1 gsc sample v2 gim sample v1 gim samples v2 gsp sample v1 gsp samples w1 x1 gsc samples v1 gim samples v1 gsp E samplet w1 x1 gsc sampled v1 gsp File name Open Files of type Loadable Images and Matchsets Cancel EA Fig 2 9 Open dialog box In the PDQuest Open dialog box Already open images are marked with an asterisk File types and icons are listed next to each image name MatchSets are listed as single files even though they are really collections of In the Open dialog box open an image by double clicking on its name or selecting the file name and clicking the Open button Specify the file type by selecting one from the Files of Type pulldown menu Your options include Loadable Images and MatchSets Raw Image Filtered Image or Gaussian Images MatchSets or All Files To select multiple files to open use the standard ctrl click and shift click key combinations in Win
37. The lens filter must be INSTALLED if you are using the fluorescent spot cutter in fluorescent and visible modes The lens filter 1s not required if you are using the spot cutter only in visible light mode Click the Calibrate Cutter button A series of dialog boxes will guide you HT Step 1 The second dialog box prompt for flat field calibration requires that you ensure the stage is completely empty and dry This includes the cutting mat which must be removed Press Ready to proceed If you have previously performed this step you can skip to Step 2 although this is not recommended 8 7 PDQuest User Guide i POQUEST X Spot Cutter Calibration step 2 Image Field Calibration This step requires the calibration template grid pattern supplied wath this system The grid should be placed on top of the cutting mat which should be installed on the cutter first Make sure the grid is flat and aligned wath the stage edges nce you have placed the grid on the cutting mat press Ready to proceed If you have performed this step before you may skip to Step 3 although this is not recommended Read Skip Back Cancel Help Fig 8 4 spot cutter calibration step 2 Step 2 The third dialog requires the calibration template grid pattern supplied with the PDQuest system Place the grid on top of the cutting mat which should have been previously installed on the cutter Make sure the grid is flat and aligned wit
38. X Cancel Help Fig 6 14 Select a sample 6 18 Analysis Tools If the sample you wish to add is not in the list click New to create a new sample This opens the Edit Sample Dialog il Edit Selected Sample a aaa x Mame Description Individual Date 15 Ju1 2002 09 50 yf Done X Cancel 21 Help Fig 6 15 Creating a new sample Enter all pertinent information for the sample and click Done The new sample is added to the list You can also import samples from from a text file by clicking Import in the Enter Sample Attibutes window This opens the Select a File dialog Locate the file you wish to import and click Open The samples and any attributes attached to the samples are added to the sample list The configuration of the file you import 1s important Sample names and attributes including values must be separated by a comma The text file should resemble the following example sample1 temp 30 deg dilution 2 fold color red sample2 temp 37 deg dilution none color blue sample3 temp 42 deg dilution 2 fold color neg sample4 temp 45 deg dilution none color no color The sample name must come first followed by the attributes In the example given the attributes are temp dilution and color Each attribute is then given a specific value for each sample For example Samplel contains the attributes temp dilution and color with the values for each being 30 deg 2 fold and red Adding Attributes O
39. and be generous with borders You can always crop the image later F 7 PDQuest User Guide F 4 Step Ill Select Resolution To select from a list of possible scanning resolutions click on the Select button under Step HI Select Resolution This will open the Select Scan Resolution dialog box i PDQUEST xl A 36 3 x 36 3 alo rx 425 fe 635 x 63 5 303 ao select Scan Resolution High For small gels with tiny features Done Medium high For gels with small features Medium For most gels Cancel Medium low For gels with large features 1247 0 s 127 0 Low For gels with very large features H Help Fig F 7 Select Scan Resolution dialog box Available resolutions are listed from highest to lowest in terms of the dimensions of the resulting pixels in microns Smaller pixels equal higher resolution Each resolution is listed with its typical use In general the size of your pixels should be one tenth the height of your smallest object Specifying Your Own Resolution If you select Oversample in the Options dialog box you can specify your own resolution within the range of 32 169 micrometers With Oversample selected enter values directly in the fields next to X resolution and Y resolution in the main acquisition window F 8 Appendix F GS 800 Step lll Select Resolution X resolution so 0 Microns Select Y resolution so _ ol Micron Image file size 0 10 ME Fig F 8
40. ellipses With the Gaussian image loaded you can also display the crosshairs and ellipses in the Filtered image and Raw 2 D Scan in the same scanset Ez Show Crosshairs F5 Spots menu or main toolbar is used to display the crosshairs in a subwindow Use the tool or the key command in the subwindow to toggle the display In a scanset the Gaussian image must be loaded To display crosshairs in all subwindows select Show All Crosshairs Shift F5 Spots menu Detecting and Editing Spots Note Spots without crosshairs in the Filtered and Raw 2 D Scan images have not been detected and are not included in the Gaussian image ul Scanset demol demol l T Wodifiecd demol vl ea Fig 4 9 Examples of crosshairs and ellipses in a scanset Show Ellipses Alt F5 Spots menu shows the shape of each spot generated by Gaussian fitting Use the tool or the key command in the subwindow to toggle the display Show All Ellipses Ctrl F5 Spots menu shows the spot ellipses in all the subwindows Hide Overlays F4 main toolbar will hide the crosshairs and ellipses on the image Use the tool or the key command in the subwindow to hide the overlays 4 5 Adding and Removing Spots If spot detection has missed some spots or identified too many spots in a cluster you can add and remove spots in the Gaussian image 4 15 PDQuest User Guide Note You can add and remove spots in the MatchSet and or the scanset Note that after a Match
41. exposures The exposures will appear in the Focus window at the rate of about 2 per second At the top of the dialog click Auto Expose to reset the exposure time of the camera The Time field shows the time of each exposure in milliseconds The Ceiling field shows the highest pixel value in the focused region The Levels field shows the levels of gray scale in the image All of these settings are automatically determined by the software based on the position and aperture of the camera lens 9 11 PDQuest User Guide Manually adjust the camera lens and position as described in the hardware manual to optimize the image When you are satisfied with the image and the levels of gray scale click Stop to close the Focus dialog Set Cutting Tip Height drops down to cut a spot The adjustment dialog box will open and the P Click the Set Cutting Tip Height button to adjust the distance the cutting tip cutting tip will move to the center of the gel Click Lower Tip to drop the cutting tip to 1ts lowest position In this position manually adjust the cutting tip height as described in the hardware manual until it presses lightly against the surface of the cutting mat Raise Tip raises the cutting tip Test Cut performs a test cut in the center of the gel Click Done to accept the settings and return the cutting tip to home position The cutting tip height only needs to be set at installation and checked every few months depending on
42. level capture an image then adjust the level and capture another image For example if you select Low Sample Intensity and the resulting image has too many saturated pixels you will receive a warning message Simply change the setting to Medium Sample Intensity and rescan If you select High Sample Intensity and the resulting image is too faint select Medium or Low and rescan Custom Applications If your application is not listed if you want to use user installed filters or if you want to use an external laser you can create and save your own custom application From the application tree select Custom then Create This will open a dialog box in which you can name your application and select your settings A aneS Chemiluminescence Chemifluorescence Create a new Imager Fx Application DNA Stain Gel Name Custom Application 1 Protein Stain Gel Fluorophores Filter A Blank Microtiter Plate Filter B Bio Rad Radioisotope Densitametry faz Elocki Custom Filter C rm Blocking Create Laser 532 1064rm PMT Voltage Low Sample Intensity Cancel OF Fig J 5 Creating a custom application J 8 Appendix J FX To select a filter including user defined or filter combination click on the buttons for Filters A B and C and make your choice from each pop up list Note Filter wheel C is not available in the FX Pro or the FX Pro Plus systems Note The user defined fi
43. or slides The scanning dimensions in this mode are 29 cm X 40 cm F 6 Appendix F GS 800 F 3 Step Il Select Scan Area Preview Scan Before selecting the particular area to scan you can preview the entire scanning area to determine the exact position of your sample Click on Preview Scan A quick low resolution scan of the entire scanning area will appear in the scanning window Your sample should be visible within a portion of this scan Selecting an Area Using the preview scan as a guide select your scan area by dragging your mouse within the scanning window The border of the scan area you are selecting will be marked by a frame Note The scan area you select must be at least 1 cm wide When you release the mouse button the border changes to a dashed blue line indicating a selected area e To reposition the scanning area box you have created position your cursor inside the box and drag The entire box will move e To resize the box position your cursor on a box side and drag The side you have selected will move e To redo the box entirely position your cursor outside the box and drag The old box will disappear and a new box will be created You can also select the scanning area by entering coordinates in the appropriate fields Top Bottom Left Right As you enter a coordinate the position of the scanning area box will change accordingly When selecting be sure to include the entire area of interest
44. select a high exposure time initially or use the Optimize Exposure command described on page 12 Preview For shorter exposures you can use Preview to test different exposure times Click on the Preview button to create a preview exposure and display it in the acquisition window Note The camera on the VersaDoc must be at the correct operating temperature before capturing images The temperature adjustment can take several minutes after the camera is turned on depending on your VersaDoc model and the ambient room temperature See your hardware manual for details A preview scan takes only half as long to create as a real scan because the preview scan does not capture a dark image The progress of the exposure will be displayed in the Exposure Status bar at the bottom of the dialog box You cannot save preview scans If you want to stop a preview scan that is in progress click on the Stop button K 5 Acquire the Image You can acquire a single exposure for each channel based on the time selected in Step III Set Exposure Time or take a series of exposures for a particular channel over a specified interval Optimize Exposure Illumination Flat Fielding For applications using the UV or white light transillumination you should use the appropriate reference plate to ensure a uniform intensity in the image This will compensate for normal variations in image pixel intensity that occur with a transilluminating light source
45. the same directory as the scanned image Windows backup files will have an sbk extension Macintosh backup files will have the word backup after the file name Note To open a Windows backup file you must first change the file extension To locate the file after changing the extension select All files in the Files of type drop down list of the Open dialog K 6 c Imaging Area Size The imaging area is the area of the sample in centimeters that is captured by the camera and displayed in the scan window To specify the size of this area enter a dimension in the appropriate field under Imaging Area in the Options dialog box Click on the Options button in the acquisition window to open this dialog When you change one imaging area dimension the other will change to maintain the aspect ratio of the camera lens The imaging area will change depending on your zoom factor For example if you have zoomed in on a area that is 4 5 x 3 5 cm then you would enter 4 5 for the width 3 5 for the height would be calculated automatically K 17 PDQuest User Guide Note Your imaging area settings must be correct if you want to do 1 1 printing They must also be correct if you want to compare the quantities of objects e g using the Volume Tools in different images The imaging area dimensions also determine the size of the pixels in your image 1 e resolution A smaller imaging area will result in a higher resolution K 6 d Auto scale
46. window The saturated spots will appear highlighted and the number of saturated spots will be listed at the bottom of the window 4 23 PDQuest User Guide You can also use the Highlight Saturated Pixels in the Transform window to identify regions of saturation in the image Faint Spots Faint Spots on the Spots gt More Edit Tools submenu and Edit Spot toolbar highlights those spots whose signal falls below a user defined threshold Select the command from the submenu or toolbar then click in the image The faint spots will be highlighted in the image and the number of faint spots will be listed at the bottom of the image window 4 10 Spot Quantity Spot Quantity is the total intensity of a defined spot in a gel image This corresponds to the amount of protein in the actual spot in the gel see section 1 2 Digital Data and Signal Intensity For spots defined using the boundary tools section 4 6 Spot Boundary Tools this is simply the sum of the intensities of the pixels inside the boundary For Gaussian spots it is calculated during spot detection and Gaussian fitting The formula for calculating the quantity of Gaussian spots in PDQuest is Spot height 1 o oy where Spot height also known as peak value is the peak of the Gaussian representation of the spot It is measured in ODs or counts image units depending on the type of imaging device used Note One image unit IU 100 micrometers or 0 1 millimeter T
47. 1 Identity Noze Characteristics Study the density distibution histogram below and select the shape vou see x Pepper x Salt Look at the density distribution plot for a background region of the image and select the shape of the plot you see Gaussian 7 Gaussian outlier Uniform ff Outlier Density Distribution Histogram Step 2 Select Filter Size Select a size in pixels that ts larger than the average noite feature fe Ses f 5x5 ot ee xa Filter selected Median 3x3 OF Cancel Help Fig 3 22 Filter Wizard dialog box The Wizard contains settings for identifying the different types of noise in the image It also includes a density distribution histogram of the noise in the image to aid in filter selection Step l Identify Noise Characteristics The first step in the Wizard is to identify the type of noise in your image Examine both the image and the density distribution histogram then select one both or neither of the following checkboxes e Salt This type of noise appears as specks that are lighter than the surrounding background The density distribution histogram of this type of noise displays 3 34 Viewing and Editing Images noise peaks at the high end of the range right end of the plot This type of noise is common in electronic cameras with malfunctioning pixels It can also be caused by dust or lint in the imaging optics or scratches on photographic film Salt is
48. 3 4 and 5 Boolean AND Boolean OR Fig 7 7 Venn diagrams depicting the AND and OR boolean operations To create a Boolean set select Boolean from the Select Set Type dialog 7 18 Analysis Sets and Annotations i Analysis Set Template Matchset HalfStock Ae a x Description x Mark spots ae A mta 579 spots Add set _ Display selected sets B qual 173 spots Remove set Compare Mark spots on e Master gel Operand gels All gels Show x window labels 1595F number f In and not in E 0 Intersection Not in and in E oo Union cD ln only A or only in B Format Spot count Be yf Save X Cancel Z Help Fig 7 8 Boolean Analysis Set Enter the name and description to be given to the analysis set In the Compare section add at least two analysis sets to use in your analysis If you choose more than two sets then you will only be able to choose between spots present in all sets or spots unique to all sets All other choices will be inactive Select the Boolean operator in the Method section When you click Save the new analysis set appears in the ASM and the spots that meet the criteria that you specified will be highlighted 7 19 PDQuest User Guide As with all other types of analysis sets when the Analysis Set Template is open you can view the results in the MatchSet immediately To the right of the dialog you can specify the spot marker
49. Alt and Reports gt Graph Retreat Alt commands to shift between regions Or you can navigate to the View menu and select the graph toolbar Erratic Spots Erratic spots are matched spots whose quantities vary significantly between member gels To mark these select Mark Erratic Responses from the Match gt Edit Matches submenu and click in the Master Enter a fold change factor in the pop up box For example a change factor of 3 will highlight spots whose quantity varies by a factor of 3 or more among member images When you click Apply the selected spots will appear circled in the Master Graph Erratic Responses displays histogram graphs of each erratic spot in the Master Select the command from the submenu click in the Master enter a fold change factor in the pop up box and click Apply to display the graphs 5 32 Matchsets Histogram bars reveal a three fold variation in spot quantity between gels SSF 1301 Fig 5 19 Graph Erratic Responses each bar represents the spot s quantity in a gel Note Histograms can only be displayed for one region of an image at a time Use the Graph Advance Alt and Graph Retreat Alt commands to shift between regions 5 8 a Colony Matching If you have a MatchSet containing colony plate images you can match colonies quickly and easily using the Colony two point Match command on the Match gt Edit Matches submenu Unlike the regular matching function you
50. As you drag the arrow will rotate and the angle in the dialog box will change Position the arrow so that it points in the direction of the new top of the image You can fine tune your rotation as much as you like 3 31 PDQuest User Guide Note If you want to center your arrow on a particular point on the image e g to align along a particular lane you can use the F3 key command Position your cursor on the point on the image you want to center on and press the F3 key The image will move so that the center of the arrow and your cursor point are aligned To complete the rotation click the Rotate button in the small dialog box A subwindow will open containing the rotated image You will then have the option of renaming your new image and changing the version number If you are not satisfied with your rotated image simply delete it and start over Note Because an image is composed of square or rectangular pixels Custom Rotation has to perform some minor smoothing on the image to turn it at a non 90 angle In addition any analysis performed on the image cannot be rotated and will be lost 3 15 Filtering Images Filtering is a process that removes small noise features on an image while leaving larger features like data relatively unaffected A wide range of filters are available for removing different types of noise from images Depending on the nature of your data you will probably need to use only one or two of the
51. Calibrate Mow Edit Step Tablet Data Handling The Densitometer will be told to scan at its maximum resolution and spatial averaging will be used to produce the desired resolution This gives better spatial fidelity and lower noise although it takes longer to scan _JOverzample A modified logarithmic scaling system will be used to reduce the size of the data by 50 It introduces a constant relative error of less than 3 TETIT Cancel Help Fig F 9 Densitometer Options dialog box Note that calibration is always on for the GS 800 F 5 a Step Tablet Values The built in transmissive and reflective step tablets on your densitometer have specific density values Before scanning for the first time you must enter the values for your transmissive step tablet into the software You can use the default values in the software for the reflective step tablet Transmissive Step Tablet Before you scan in transmissive mode for the first time you must specify the values for your transmissive step tablet First make sure that the Transmissive checkbox is selected in the main acquisition window then click on the Options button In the Densitometer Options dialog click on the Edit Step Tablet button The Step Tablet Values for GS 800 Transmissive dialog box will open F 10 Appendix F GS 800 334 Quantity Offset 0 000 a Step Diffuse Specular Step Diffuse Specular 1 0 05 o 07 12 1 75 2 47 2
52. DOES po a a las ias a a 5 26 COS egino o a ES 2 15 Crean 4 ia a ENT A TE E 5 3 Exporuns di ua a a ts 2 25 Hio lere aeaa E abas cen iouet sueeaes set besw sa Ceeetetan sa Ain ieannsmaaetnaieate 5 34 PP A O 5 17 A E glee mews nian R 2 10 Removins alll matches sust 5 33 SAVINO tot died Ocio ea 2 16 Undone matones scaretoatecnandicscescoscssat e E EE OAN 5 30 Memorvallowande ennonn irran a a E E 2 35 MEDUS as 2 1 Mersine multiple E xpOSUr S main is L 10 Molecular Imager FX NC CUM CNG nato arisia teas E a tide suse sleds adas deals J 13 COTE OATS laa cil J 2 Filesize ot Image S dl sa ee ets eae aan teenies J 13 CDOS ai sacras A Peas PU A J 14 Saturated pixels highlighting aaa o J 14 Savino tas Maso tol J 13 SCAN MANOS J 3 SElSCUNS AN APPlIC AON airia EO E E EE J 4 SELECUNS roo blo eian E a Ai J 12 Ai aa a a Sagaondaa ics aabaanels J 11 SIMAO mode odds J 2 INIGUSE2AS STON AIC roO l Sissonne a eercognedasdasouatcaiascarebladevbapshuntentauhiactincdes 2 45 Behavior with keyboard commands ccccccssessessssesseeeceseceeeeeceeeeeeeeeeeeeeees 2 6 MED DA enr a sico 6 29 DN tere hee tiene E A 6 30 FIMO Oi 0 4 scoadcensensiea simbolos doi E coccion 6 30 Index 12 Transferring in high level MatchSets ccccccseessessseseseeeceeeeeeeeeeeeeeeeeeeees 6 31 Molti hanne VIEW uyana a 3 16 Normalization vonga ais 6 23 Open From Works Bast renina a ot N 9 Opening TES O RES 2 10 Optimizing images see Transform Overlays M
53. Done 7 36 Analysis Sets and Annotations Printing Annotations for All Spots All Spots Specify Categ s prints selected annotation categories for all the annotated spots All Spots All Categ s prints the annotations in all available categories for all annotated spots 7 6 Transferring Annotations If a member of a higher level MatchSet includes annotations you can transfer all or some of them to another higher level MatchSet member or to the higher level MatchSet Master Note Only annotations for spots that are matched in the higher level MatchSet will be transferred If no spots are matched between the members only categories will be transferred With the higher level MatchSet open select Transfer Annotations from the Analysis gt More Annot Tools submenu In the dialog box select the member MatchSet that you want to transfer from using the From MatchSet pulldown list 7 37 PDQuest User Guide hi Transfer Annotations Ea From Mabtchset mat chset demo Total spots C d To Matchzet Matchset demoxx Total pots ass Category All categories Specified category Matched spots 438 x Replace categories x Confirm before replacing yf Done X Cancel z Help Fig 7 20 Transferring annotations Select the member MatchSet that you want to transfer to using the To MatchSet pulldown list Note that when you select the second MatchSet the total spots in each M
54. E AAA 8 3 9 11 TN 8 4 9 12 O 8 2 9 11 SpoL Detection WiZard sasea oae aaO E a E a AS G 4 1 Backsround subtraction sessions 4 7 PINGING SPOL Centers cnica daci n 4 4 Identifying faint small and large spots ooocccccccccncnonononononnnnnnnnnnnnnnonnnnnnnnnos 4 3 INOISE TOMO Vals Xaros sacra doracancsssncnseveniat EE ERE O 4 7 Parameters loa dino sitas lolis 4 8 PROCES SIO IS is 4 9 Savine paramo ad co 4 8 Sensitivily and SIZE Scale eines porros 4 5 SRG Ale TC 001901 PPP ORO PA 5 OO aces ni teaeNstisadausautese aust sade 4 7 SPOL ELIPDSES ada E 4 14 Spot Review LOOM ius rl chidas 6 1 Spots AIGE MOON OO SO O a a a a 4 16 Boluda violation 4 18 A cess amen aeese ahaa cen io etc EAE 4 22 CONTIN alioli saaenqnee oaanoonstcetgutimeb ied E 4 23 CONDUTO ci ctl e An A 4 19 TOSS ATES nl lo Ss 4 14 PR tee Tare Teeny arene en ry Senet eemeTy ener Teen 4 9 Detection parameters SCICCHNS aiii leidas 4 1 Detection report seso eo io cle alcoi ads 4 11 Peteca on Wizard cunas 4 1 A bate ean sa Oeaotioeinns ea niaieannsnaeetnaneatea 4 14 A te dont AA E E neces tencadea sims A 4 24 Index 16 PWG A e E A OOO A eaieaseeaaabens 4 28 Prechand 100l rs a 4 20 TOU ality sets atada idas 4 27 Low guany el S alo id 4 25 No A cr ere tered 4 28 A OES aren TA cacao T OR 4 28 Pear all le rte E EN EE A E N 4 28 O 4 26 4 28 QUAN loa ne 4 24 4 28 A A A 4 26 A duvet Coden hago tment E O oa Coens 4 17 FSC VIC We LOO I E E T AE AT E E A A T E ATAT
55. Entering a custom resolution with Oversample selected Image File Size The size of the scan file for the selected resolution is listed next to Image File Size If you do not have enough computer memory for the specified file size an error message will appear when you attempt to scan If this happens select a lower resolution or decrease the size of the area to be scanned Macintosh users can also increase the application memory partition See your Macintosh computer documentation for guidance F 5 Calibration The GS 800 automatically calibrates the densities of scanned images to ensure accuracy and reproducibility of results The GS 800 has built in step tablets for both transmissive and reflective scanning e The transmissive step tablet calibrates transmissive scans from 0 to 3 0 O D e The reflective step tablet targets reflective scans from 0 to 2 0 O D The first time you use the GS 800 you must select some settings to ensure accurate calibration Click on the Options button in the main acquisition window This will open the Densitometer Options dialog box F 9 PDQuest User Guide Densitometer Options On the GS 00 this requires that the calibration overlay be installed Calibrate Before Every Scan Recalibration The Densitometer will be re calibrated Su Interval minutes after this time has expired ap A calibration report will be printed each time the Lj Calibration Report Densitometer is calibrated
56. Exporting an Image To export a Raw 2 D scan Filtered image or Gaussian image as a TIFF image select Export to TIFF Image from the File gt Export submenu then click the image in a MatchSet or scanset Analysis export raw data Publishing e expork view excluding overlays export view including overlays Resolution dpi e 2 dpi Same az scan 139 5 150 dpi 7 Specify 22 0 300 dpi Transform Linear 7 Entire image 16 bit te Curent view 24 bit RGB Size Pizel zsa Y s1e Image 107 25 Hb EH Espot gt Cancel 21 Help Fig 2 17 Exporting TIFF Images ft Curent view In the export dialog first select whether you want to export the original image data Analysis option or the currently displayed view of the image Publishing option Note Only the Publishing option is available if you are exporting from the Mutli Channel Viewer 2 26 General Operation Analysis Export Mode The Analysis option exports the image data unmodified by any viewing adjustments you may have made such as Transform or Zoom If you select this option the other controls in the dialog box become inactive Publishing Export Mode If you select the Publishing export option the exported TIFF image will look like the image as it is currently displayed on the computer screen You can choose whether or not you want to include overlays Specify a resolution for the expor
57. Raw 2 D Image A samples vl Gaussian Fig 5 2 Example of a MatchSet The Master is in the upper left subwindow Note While MatchSets can contain gels with different quantitation units quantitative spot comparisons can only be made between gels that have the same units 1 e Matchsets spot quantities measured in O D s cannot be compared to spot quantities measured in counts Qualitative comparisons are still valid 5 1 Creating a MatchSet A MatchSet is created from the Gaussian and Filtered images as well as the original 2 D scan of the gel s in an experiment After you have completed gel selection and spot detection in the Automated Detection and Matching dialog just proceed to Step 3 Creating a MatchSet To create a MatchSet later select New MatchSet from the Match menu PDQuest User Guide 5 1 a Create a MatchSet I Create MatchSet on disk iA x Name New Match Auto Description Comments rMatchSet level Member sharing Level 1 members are gels Do not make copies refer to the originals Higher level members are MatchSets Copy the originals into this matchset s data folder gt Members Add f sample1 v1 398 spots sample2 v2 260 spots EX Remove sample3 v1 298 spots sample4 v1 282 spots 003 Master samples v1 305 spots sample6 1 331 spots Count le i Drag items to reorder them Save X Cancel Help
58. Remove MiIPLD AA a a 6 30 AAA A sSiodaensastiateees L 9 RemOvIN poetes 4 17 Replicate Group EHEC vo bho CAPARO O PO E PE ice E te ttene en meer arrerr Ter eey err tee vrerr 6 10 REM o El 6 10 Reports AaB COMPAS OM saaa a a a 11 7 Normal ATION co aa 6 23 Quantitati n Table sirarne aaa TA N anak 11 9 CATO eragon E E alae 11 9 Quante Ora PIS aen a e N 11 11 callen PIOUS salta 11 14 Standard Maserati 11 13 Reve OS aV e oases soni ascmnsneneanheapnonarassaiansarmacedaacaue E lanehiniend pierre 2 16 FG CGM ATM AS S eao E Nine renee E E N 3 29 Sample Database nta in 6 17 Sample IMA a A A A 2 12 Sample PAIS seas 6 21 Saturated pixels Menio tna litis 3 26 See also individual imaging devices SAGE ALS CS IO A a 4 23 Save a le edo delta 2 16 Saving Existing Files in WorksBase vcd N 14 Scans PADUNE A A OS 2 21 SCA sde 4 12 A dos 2 8 Seater Pl OM MOO ad cd 6 7 SBOW MAA a 5 22 Simulation mode see individual imaging devices SoOMWware cesado 1 13 SPOLMOUNCAICS a A ol asisenianasoaeaasteasuane 4 18 Index 15 PDQuest User Guide COOU as rs A reeane een mere re 4 19 Cop yim and Pas tins ad ln 4 22 Dele TO teus tit 4 21 Freehand TOOL setas 4 20 A A yet freer etre tem 4 21 SDOL CLOSSIAIES Jog icsaentectins lent osscus tah a el tansaetiadtatisahae mates 4 14 Spot cutter ISAS TC ii leerla noia 8 1 Calor OO alaba 8 6 9 15 CUT OUI OMS si ld acido load 8 16 GUUS TID EIS AU etorri 8 3 9 12 FOCUSSING the Camera attics ese aae ar a A
59. Section 6 6 d Sample Pairs Step 5 Once you have created your sample pairs go to the Analysis Set Manager Click create and select Statistic from the Select Set Type dialog Analysis Sets and Annotations Step 6 Choose to compare Classes and select the select the classes by clicking on the drop down buttons for A and B The Wilcoxon test is now available for you to use in your analysis Click the Table button to view a table listing degrees of freedom and critical values Click Save and the new analysis set is added to the ASM with significant protein spots highlighted in the MatchSet As with all other types of analysis sets when the Analysis Set Template is open you can view the results in the MatchSet immediately To the right of the dialog you can specify the spot marker on which gel s to show the spots and what information to show on the gel s When you open the Analysis set Template whatever analysis sets you have selected in the ASM become inactive To view these sets in the Analysis set Template check the box labeled Display Selected sets 7 1 f Arbitrary Analysis Sets Arbitrary analysis sets are composed of any group of spots that you choose To create an arbitrary analysis set select Arbitrary from the Select set type dialog 7 15 PDQuest User Guide i Analysis Set Template Matchset HalfStock ae 0 x Mame JArbit Description gt Mare spots x Operand Member Display selected sets
60. See the hardware manual for more details PDQuest User Guide PC Only A Note About SCSI Cards The Fluor S is connected to your computer by a Small Computer System Interface SCSI cable To use the Fluor S you must have a SCSI card installed in your PC If you have a PC with a Windows 98 or Windows ME operating system you may also need to load the SCSI and WinASPI drivers that came with the card Simulation Mode Any of the imaging device acquisition windows can be opened in simulation mode In this mode an acquisition window will open and the controls will appear active but instead of capturing real images the window will create dummy images of manufactured data You do not need to be connected to an imaging device to open a simulated acquisition window This is useful for demonstration purposes or practice scans To enter simulation mode hold down the CTRL key and select the name of the device from the File menu The title of the acquisition window will indicate that it is simulated G 1 Fluor S Acquisition Window To acquire images using the Fluor S go to the File menu and select Fluor S The acquisition window for the imager will open displaying a control panel and an image display window Appendix G Fluor S Fluor S Multilmager Step Select Application Select Silver Stain Clear White Trans HiRes Scan Dimension 80mm 160mm 250mm 300mm Step I
61. Serial Number pulldown list Next select the plate used for the cut run using the Microtiter Plate pulldown list If more than one plate was used for the run select the first plate you want to process step 2 Select the appropriate Micromass instrument using the MALDI or MS MS option button Step 3 The Required Project Name is the name of the MassLynx project that must be created for this cut list Open MassLynx and create a project with this name See MassLynx documentation for information on how to create project files Note We recommend that you create a MassLynx project file from an existing project or a default template to ensure that the default parameter files are stored in the correct location inside the project When you have created a project you can click the Find button if the path to the project directory is mapped correctly as described above or navigate to it yourself by clicking on the MassLynx Project pulldown button Step 4 In this step you specify the mass spec parameters to be used for processing the proteins See Micromass documentation for descriptions of the parameters and their functions Note You must map the Micromass paths as described above to specify the parameters Select the acquisition parameters using the Acquisition Parameters pulldown list Select the processing program you want to use using the Process With pulldown list 10 2 Mass Spectrometry Analysis Select the processin
62. The new analysis set appears in the ASM and the spots that meet the criteria that you specified will be highlighted As with all other types of analysis sets when the Analysis Set Template is open you can view the results in the MatchSet immediately To the right of the dialog you can 7 21 PDQuest User Guide specify the spot marker on which gel s to show the spots and what information to show on the gel s When you open the Analysis set Template whatever analysis sets you have selected in the ASM become inactive To view these sets in the Analysis set Template check the box labeled Display Selected sets 7 2 Annotation Tool You can add notes comments and other pertinent information to each protein spot in a MatchSet using annotations Annotations can be used to describe spots group them into categories and reference additional information in other files or Internet databases You can import MassLynx mass spec data into annotations Spot annotation data is automatically stored in HTML format for easy portability and uploading to Web sites 7 2 a Opening the Annotation Tool Spots are annotated using the Annotation Tool The tool can be opened by selecting a spot or spots in the MatchSet or you can open the tool with no spots selected 7 22 Analysis Sets and Annotations x r Select Category Select Spots gt r nnotation Entry a
63. Tip Height 7 Click the Set Cutting Tip Height button to adjust the distance the cutting tip drops down to cut a spot The adjustment dialog box will open and the cutting tip will move to the center of the gel PDQuest User Guide Click on Lower Tip to drop the cutting tip to its lowest position In this position manually adjust the cutting tip height as described in the hardware manual until it presses lightly against the surface of the cutting mat Raise Tip raises the cutting tip Test Cut performs a test cut in the center of the gel Click Done to accept the settings and return the cutting tip to home position The cutting tip height only needs to be set once unless the spot cutter gets out of alignment 8 1 c Spot Cutter Settings Click the spot cutter Settings button to open the settings dialog box for the Feb cutter i Spot Cutter Settings E x Cutter model Not set Froteomew orks Spot Cutter 25cm f ProteomeWorks Plus Spot Cutter 30cm Microtiter plate Well count e 96 wells 7 384 wells Loading order fe A 42 43 41 81 C1 Wash station 1 position o o 0 0 Miscellaneous Cutting tip f 1 0mm 1 5 mm diameter Crop left margin a Done X Cancel 2 Help Robot e COM 1 COM 2 Fig 8 3 spot cutter settings 8 4 Basic Excision Tool Cutter Model Select the spot cutter model you will be accessing for your cut run This setting most lik
64. Tool would load your spots from one MatchSet analysis set on the plate then select a different MatchSet analysis set and skip the previously loaded wells The Current Run Serial field displays the serial number of the cut run The cut run serial number increments whenever you perform a previous cut run on a new MatchSet gel or gels For example if you first cut spots from one gel in a MatchSet and do a cut run then cut the same spots from a different gel in the MatchSet and do a run the serial number for the second run would be 2 9 2 a Spot Cutter Setup Before you can use the spot cutter for the first time you must focus the camera and adjust the cutting tip height You must also adjust the spot cutter settings and calibrate the instrument Note If you have already focused and calibrated the spot cutter using the Basic Excision Tool you do not need to perform these steps again Focus Cutter Camera Note If you are using a lens filter make sure that it is installed before focusing If you add or remove the lens filter after focusing you will need to refocus The Focus Cutter Camera dialog box controls the camera exposure setting Focusing and aligning the camera are described in the spot cutter hardware manual y Click the Focus Cutter Camera button to open the dialog The platform light on the spot cutter will turn on and the camera will focus on a small region at the center of the gel or membrane and begin taking continuous
65. Under Spots to Export specify the spots whose data should be exported You can choose all the spots in the MatchSet those spots that are matched in every member of the MatchSet or the spots in a particular analysis set The spot count automatically adjusts to reflect the total number of spots selected Under Output Options you can include headers for each column in the report Select how you want to sort the spot data by SSP number DSN or gel replicate group Select the field separators you want to use to separate columns of data in your spreadsheets commas or tabs for Excel Your data can be exported as a file or to the clipboard If you choose the file option button clicking Done opens the Export MatchSet Data dialog box in which you can enter a name and export location for the file The file type automatically defaults to an xls spreadsheet If you choose the clipboard option clicking Done saves your data to the system clipboard If you have created replicate groups for the MatchSet you can export the averaged data for each group Under Spot Data by Group select the data to export Master position average quantity coefficient of variation standard Gaussian size average normalized quantity normalized coefficient of variation and or the counts in the member gels 2 30 General Operation Data to export Spot data by gel Spot data by group Master position x 4vg quantity Coeff of variation x Master gaus
66. User Guide Step 4 Align Analysis and Cutter Images After you have selected the cut list and the cut run settings click the Align button gt xi Please place gel samples on the cutting stage and click Gel Ready to proceed Gel Ready Cancel Fig 9 8 Place gel on spot cutter dialog You will be prompted to place the first gel in the cut list on the spot cutter platform Click Gel Ready when you have positioned the gel as shown in the image The Align Analysis and Cutter Images dialog box opens The process of auto alignment consists of the following steps Step 1 Acquire the image of the gel with the spot cutter camera Step 2 Position the data area box in correct position 9 22 Integrated Excision Tool Step 3 Perform alignment In Step 3 you will have the option of skipping auto alignment by making the software work in totally manual mode This could be useful if you have very few spots to be cut so you can do the alignment yourself Step 4 Set the confidence threshold value Visually confirm and if needed manually correct the alignment of all those spots whose alignment is below the confidence threshold i Align Analysis and Cutter Images stock1 1 Filtered Cutter Camera Image Eee bab Step Image Acquisition Step 2 Data Box Placement Step 4 Alignment Confirmation Display Options hiAcquire image E Reposition X Display cropped image X Cu
67. Viewer 3 11 a Positioning the Image Windows For Windows operating systems use your mouse to reposition and rotate the image e Rotate the image Left click and drag to rotate the image e Reposition the image Right click and drag to reposition the image PDQuest User Guide e Zoom in out To zoom in or out Click the center mouse button or roll the wheel If you do not have a three button mouse or a mouse with a wheel hold down the shift key and left click and drag to zoom in or out Macintosh For Macintosh operating systems use mouse and keyboard combinations to reposition and rotate the image e Rotate the image Click and drag to rotate the image Reposition the image Ctrl gt click and drag to reposition the image e Zoom in out Shift gt click and drag to zoom in or out 3 11 b Display Mode The 3D Viewer window allows you to view the image in three different modes wire frame lighting and textured e Wire frame shows the image in a transparent frame view e Lighting shows the image with different areas of light and shadow depending on the angle of view Use the slider bar to adjust the intensity of the lighting e Texture gives the image texture Use the Scale function to scale the image This is useful for viewing shallow spots in the 3D Viewer If you lose the image because you moved it too far past the window border or rotated it and disoriented the view click Reset View to return the image
68. White Diffusion Plate Weak Chemiluminescence 5 20 min 50 mm Chemi Tray if sample is small Strong Chemiluminescence 30 sec 2 min 50 mm Chemi Tray if sample is small Increase exposure time two fold for each step increase in f stop For sharper focusing close the f stop down 1 2 stops from full open while focusing SFor chemi applications the 50mm lens is recommended Always remove the 660 filter For most applications you can select an exposure time capture an image study it then adjust the exposure time accordingly Repeat this procedure as many times as necessary to obtain a good image For chemiluminescent samples which degrade over time and emit low levels of light you can select a high exposure time initially and use Live Acquire mode to save intermediate exposures see following section Preview For shorter exposures you can use Preview to test different exposure times Click on the Preview button create a preview exposure and display 1t in the acquisition window PDQuest User Guide A preview scan takes only half as long to create as a real scan because the preview scan does not capture a dark image see the following section on Options The progress of the exposure will be displayed in the Exposure Status bar at the bottom of the dialog box You cannot save preview scans If you want to stop a preview scan that is in progress click on the Stop button G 5 Acquire the Image The Fluor S
69. Zinc stain Stain all Positive dark bands light background Photograph Negative light bands dark background Coomassie stain Colloidal gold stain Amido black stain Blot AP Substrate BCIP NBT HRP Substrate 4CN HRP Substrate DAB Fig E 4 Applications available in the GS 710 acquisition window PDQuest User Guide Choosing Your Own Settings If you know the filter and light source settings you want or want to experiment with different settings you can choose them yourself Filter Red x Green jBlue hite Light Reflective e Transmissive Fig E 5 GS 710 Custom Application controls Next to Filter click on either the Red Green Blue or White checkbox or a combination of two of the first three Red Green Green Blue Red Blue Filter Color Wavelength Application Examples 595 750 nm Coomassie G 250 BCIP NBT Fast Green FCF Methylene Blue 520 570 nm Coomassie R 250 Basic Fuchsin 400 530 nm Crocein Scarlet Gray Scale 400 750 nm Silver Stains Copper Stains Film Fig E 6 Examples of filter colors and applications Next to Light select the appropriate light source e Reflective mode is for scanning opaque mediums such as dried gels on filter paper arrays or photographs The scanning dimensions in this mode are 21 cm X 29 cm e Transmissive mode is for scanning transparent mediums such as films gels or slides The scanning dimensions in this mode are 20 cm X 23 cm E 6
70. a type of outlier noise see below e Pepper This type of noise appears as specks that are darker than the surrounding background The distribution histogram of this type of noise displays noise peaks at the low end of the range left end of the plot Its causes are similar to those of salt noise Pepper is a type of outlier noise see below Next select one of the following option buttons to describe additional features of your noise e Gaussian The distribution histogram of this type of noise has a Gaussian profile usually at the bottom of the data range This type of noise is usually an electronic artifact created by cameras and sensors or by a combination of independent unknown noise sources e Uniform noise This type of noise appears in the histogram as a uniform layer of noise across the data range of the image e Outlier noise This category of noise includes salt and pepper noise see above The distribution histogram of this type of noise displays noise peaks at the high and low ends of the range After you have identified the type of noise go to Step 2 Step 2 Select Filter Size Image noise is filtered by means of a filtering window or kernel which is measured in pixels This filtering window slides across the image processing the pixels within it The available filter dimensions range from 3 x 3 pixels to 9 x 9 pixels To select an appropriate size magnify a background region of your image so that you can see
71. accordingly When selecting be sure to include the entire area of interest and be generous with borders You can always crop the image later D 4 Step Ill Select Resolution To select from a list of possible scanner resolutions click on the Select button under Select Resolution This will open the Select Scan Resolution dialog box D 7 PDQuest User Guide Quantity One A Select Scan Resolution 42 3 x 212 Highest For small gels with tiny features 42 3 x 423 High For small gels with tiny features 635 x 63 5 Medium high For gels with small features Gace 63 5 x 423 Asymmetric medium high For gels with tiny bands 84 7 x 84 7 Medium For most gels Help 84 7 x 423 Asymmetric medium For most gels with bands RE 105 8 x 105 8 Medium low For gels with large features 105 8 x 423 Asymmetric medium low For gels with large bands 169 3 x 169 3 Low For gels with very large features 169 3 x 63 5 Asymmetric low For gels with very wide bands Fig D 7 Select Scan Resolution dialog box Available resolutions are listed from highest to lowest in terms of the dimensions of the resulting pixels in microns Smaller pixels equal higher resolution Each resolution is listed with its typical use In general the size of your pixels should be one tenth the height of your smallest object Some of the resolutions are asymmetrical meaning that the resolution is higher in the vertical dimension 1 e the pix
72. all members You will be prompted for a name for the settings When matching subsequent images you can recall these settings by name Since the matching models are difficult to describe adequately in words we recommend that you explore the various matching methods on your images and find a combination that gives the best results the most correct matches with a minimal number of incorrect matches The incorrect matches are easier to find using the spot review tool than are missing matches Classic Matching By default Automated Matching occurs immediately after each landmark is placed in all the MatchSet members To turn off this feature deselect Auto match after Landmark in the Applications tab under Edit gt Preferences 5 18 Matchsets Note Most MatchSet gels require at least two landmarks before auto matching will occur The gel that was used as the template for the Master may be auto matched after only one landmark is placed If you select Match gt Automated Matching you open the Automated Matching dialog The dialog contains two tabs one for Primary Matching settings and one for Extended Matching 5 19 PDQuest User Guide Primary Matching fi Automated Matching Matchset openheart Primary Matching Extended Matching Find landmarks automatically and calculate an accurate matching _ Enable overlapped pH range matching strict i i PDQUEST E x Matching Precision 50 Matching Parameters E Maton
73. an individual spot including its quantity quality peak value location in the gel Mr pl data if any and the sigmas of its Gaussian curve Select Spot Parameters from the Spots menu then click on a spot in a Gaussian image to display the data for that spot Alternatively position your cursor on the spot and click F6 thi PDQUEST a x Spot Parameters SSP 1811A DSH n a Quantity 34611 43 center mm 22 32 127 94 slgmas mrm 0 85 0 58 peak yal 222 2E Mrpl 34 2 ele ile quality 53 OF Fig 4 17 Spot Parameters information box 4 13 Finding Spots You can find spots in a MatchSet by SSP number or DSN Select Find Spot from the Spots menu Shift F9 click in the MatchSet member or standard you want to search and enter the SSP or DSN number in the pop up box 4 28 Detecting and Editing Spots Click OK to complete the search The spot will appear highlighted and numbered on the image 4 29 PDQuest User Guide 4 30 5 MatchSets After the protein spots in your gels have been detected you are ready to compare spots across gels To do so you must create a MatchSet You can create a MatchSet in two different ways with the Match gt New MatchSet command or through the Spot gt Automated Detection and Matching dialog Step 3 The commands for editing MatchSets are located in Match gt MatchSet Tool and Match gt Edit Matches submenu A MatchSet is PDQuest s mechanism for comparing and anal
74. appear highlighted in red in the scan window and in the pop up image window To view hide saturated pixels in the pop up image window use the Image gt Transform command File Size of Images Image File Size shows the size of the image file you are about to create This size is determined by whether the image was created in High Resolution or High Sensitivity mode If you do not have enough computer memory for the specified file size an error message will appear when you attempt to acquire an image Macintosh users can increase the application memory partition See your Macintosh computer documentation for guidance G 17 PDQuest User Guide Appendix H Fluor S MAX Multilmager Fig H 1 Fluor S MAX Multilmager Before you can begin acquiring images using the Fluor S MAX Multilmager the imaging system must be properly installed and connected with the host computer See the Fluor S MAX hardware manual for installation startup and operating instructions Note Make sure that the temperature light on the Fluor S MAX is green before attempting to capture an image If you are using a PC the Fluor S MAX should be turned on and the initialization sequence completed before the host computer 1s turned on See the hardware manual for more details PDQuest User Guide PC Only A Note About SCSI Cards The Fluor S MAX is connected to your computer by a Small Computer System Interface SCSI cable To use the Fluor S MAX you must
75. automatically calculates the specular density Specular density is a measure of the light that passes directly through a medium Diffuse density includes light that is scattered as it passes through the medium Step tablet values are given in diffuse density but are measured by the scanner in specular density and therefore must be converted according to the specular diffuse O D ratio This conversion does not affect quantitation q Specular Light passes directly through medium lt q Medium gel film etc eS NX lt a Diffuse Light is scattered by medium Fig D 11 Specular and diffuse density Diffuse density values are converted to specular optical density units according to the following formula Specular OD 1 4 Diffuse OD D 13 PDQuest User Guide D 6 b Calibration Settings After you have entered the step tablet values you can immediately calibrate by clicking on the Calibrate Now button in the Densitometer Options dialog box You can also specify how often you want the densitometer to automatically recalibrate Either click on the Calibrate Before Every Scan checkbox or enter a Recalibration Interval in minutes in the appropriate field Note With calibration turned on the scanner will automatically recalibrate each time you change your filter or your reflective transmissive setting If you select a different application with the same filter and light settings it will not auto recalibr
76. average between the two extremes The ratio to which you adjust the slider is displayed in the white box to the right of the slider Select matching settings from the default settings listed in the dropdown menu Select the gel members you want to match from the list in the Matching Parameters pop up box Select the gel members you want to match from the Select Members pop up box As automated matching proceeds the numbers of Matches Extensions New Spots and Sum total display in the appropriate boxes To auto match all the gels in the MatchSet select Classic Match All Gels from the Match menu or toolbar To auto match a single gel with the Master select Classic Match Gel from the Match menu and click in the gel subwindow Or click the Match All button at the bottom of the Automated Matching dialog Click the Done button when you have completed your matches Extended Matching Use Extended Matching to_choose the method you want to use to extend primary matches Your options in the Extension method dropdown list are Disabled Restricted or Classic The color of the match symbol for extended matching is blue 5 5 a Show Symbols Match gt Show Symbols highlights all the different types of matches and or unmatched spots using different labels Select the tool from the Match menu or toolbar and click in the Master or member gel subwindow or use the F7 key command in the appropriate subwindow e Auto matched spots are labeled wit
77. be a MatchSet created from your original gels a level 1 MatchSet or it could be another higher level MatchSet Many experimental protocols can be designed to take advantage of higher level matching For example one experiment may find all the proteins that change during cell growth in culture while another may identify nuclear proteins by cell fractionation These two experiments can be analyzed individually in level 1 MatchSets each with their own Masters These two Masters could then be combined into a higher level MatchSet with its own Master Data from the two experiments can now be compared and the higher level MatchSet might shed light on growth related proteins found in the nucleus Higher level MatchSets are actively linked to the MatchSets used to create them If you edit spots in the member images of a level 1 MatchSet those changes will take immediate effect in the higher level MatchSet For example when working with a higher level MatchSet you may notice that a spot is present in one member and not the other You could open the level 1 MatchSet with the missing spot and see if that spot in present in any of the gels If it is you could add it to the Master of the level 1 MatchSet and it would appear in the corresponding member in the higher level MatchSet ready to be matched 5 34 Matchsets i Matchset mpdi00187 olx i Matchset mpdi00318 _ oy x STD mpdi00187 stdO 679 vO xO Gaussian mpdil 3686 v0 x0 Ga
78. be formatted with all spaces and underscores removed The only underscores that should remain are the item separators If you generate a Masslynx worksheet be sure to check the box labeled Format for use with Mascot This automatically formats the data file name exactly like the search title with _mass txt attached See Section 10 1 Creating a MassLynx Batch file 10 9 PDQuest User Guide Mascot Peptide Mass Fingerprint Your name G Jetson Email GJetson Protedu Search title MS142002_spots_1_Platel15303 Datahase MSDB TaxonomylAllenties H Enzyme Trypsin bs Allow up to 1 y missed cleavages Fixed Acetyl MN term Variable Acetyl N term modifications Acetyl K modifications Acetyl K Amide C term G Amide C term G Biotinylated M terrr Biotinylated M terrr Biotinylated K Biotinylated K Protein mass kDa Peptide tol 1 D Da Mass values E MH M Monoisotopic Average Data file SERIE EE ma Browse Query a HE Contents of this field are ignored if a data file is specified Ed Overview Report top 20 hits Fig 10 4 Peptide Mass Fingerprint Search dialog For example a data file created in the Generate Mass Lynx Worksheet dialog might look like this MS142002_spots_1_Platel_1_5303_mass txt Where MS142002 is the MatchSet name spots is the cut list 1 is the cut serial number plate is the plate name 1 is the run sequence and 5303 is the sp
79. checkbox selected when a scan is created and saved a backup copy will be placed in the same directory as the scanned image Windows backup files will have an sbk extension Macintosh backup files will have the word backup after the file name Note To open a Windows backup file you must first change the file extension To locate the file after changing the extension select All files in the Files of type drop down list of the Open dialog F 14 Appendix F GS 800 Highlight Saturated Pixels When this box is checked any saturated pixels in the image will appear highlighted in red in the scan window and in the pop up image window To view hide saturated pixels in the pop up image window use the Transform command on the main toolbar or Image menu Hide Grid To hide the gridlines in the scanning area window click on the Hide Grid checkbox F 15 PDQuest User Guide F 16 Appendix G Fluor S Multilmager Fig G 1 Fluor S Multilmager Before you can begin acquiring images using the Fluor S Multilmager the imaging system must be properly installed and connected with the host computer See the Fluor S hardware manual for installation startup and operating instructions Note Make sure that the temperature light on the Fluor S is green before attempting to capture an image If you are using a PC the Fluor S should be turned on and the initialization sequence completed before the host computer is turned on
80. command After you change the exposure numbers you will end up with files named RootName x1 RootName x2 RootName x3 etc Why Merge Exposures Because 8 bit scanners have a limited dynamic range dark spots may saturate on long exposures while faint spots may be missed on short exposures and either extreme can be missed on medium duration exposures Therefore it is frequently necessary to combine the quantitative data from a number of 8 bit exposures of the same gel exposed for different lengths of time into a merged synthetic image Merging creates such an image automatically as you spot detect L 10 Appendix L Techniques aa Fig L 5 Diagrammatic representation of merging Cropping and Aligning Exposures of the Same Gel for Merging Before you can merge multiple exposures you must crop and align them so that they are exactly the same size and position To do this you will use the Advanced Crop commands on the Image menu See section 3 13 a for instructions PDQuest User Guide L 12 Appendix M Cross Platform File Exchange It is possible to move image data between Discovery Series software applications on different platforms There are different protocols depending on the platform PC or Macintosh you are transferring from and to M 1 Macintosh to PC To transfer a file from a Discovery Series application running on a Macintosh to a Discovery Series application running on a PC you need to tag the file name wi
81. density in situ samples 1 536 well microplates high density arrays samples with very closely spaced bands Files scanned at 50 micrometers can be very large 100 micrometer resolution is useful for typical gels and arrays 200 micrometer resolution is useful for gels with large bands and dot blots 800 micrometer resolution should be reserved for very large objects such as CAT assays J 12 Appendix J FX File Size of Images Image File Size below Select Resolution shows the size of the scan file you are about to create If you do not have enough computer memory for the specified file size an error message will appear when you attempt to scan If this happens select a lower resolution or decrease the size of the area to be scanned Macintosh users can also increase the application memory partition See your Macintosh computer documentation for guidance J 5 Acquire the Image Once you have selected your application scan area and resolution you are ready to acquire an image Click on the Acquire button There may be a short delay while the image laser warms up then the scanned image will begin to appear in the scanning window line by line To interrupt a scan click on the Stop button A message will ask you to confirm the interrupt and then you will be asked if you want to keep the partial scan This feature is useful if you overestimated the size of the area you selected Note Ifthe image you are scanning has more
82. disables the remaining controls in the dialog box the software will examine the data range in every image and optimize it accordingly Auto All will auto scale all the images in the selected scanset or MatchSet and close the Transform dialog High Low Sliders Use the High and Low sliders to optimize the image manually Drag the High slider handle to the left to make weak signals appear darker Drag the Low slider handle to the right to reduce image background As you drag examine the Frequency Distribution histogram Everything to the left of the low slider will be remapped to minimum intensity while everything to the right of the high slider will be remapped to maximum intensity Position the markers at the ends of the data range in the histogram and use the low slider to cut off the spike of background noise You can also type specific high and low values in the text boxes next to the sliders Clicking anywhere on the slider bars will move the sliders incrementally If your image data is in a narrow range as shown in the histogram select the Log High Low Sliders checkbox This allows for finer adjustments with the slider handles 3 23 PDQuest User Guide Frequency Distribution f Full scale Low High Log Range of actual data in the image is very limited Low slider remaps background noise to white High slider remaps weaker signals to black Low High magnifies Log scaling enables you to Can better distingu
83. entered data The second option displays MrpI values in red next to the spots 6 30 Analysis Tools For a graphical display of the overall trend of MrpI values across the entire gel first make sure the Master subwindow is active then select Plot MrpI Grid from the Analyze gt MrpI Standards submenu or toolbar Enter Mr and pl e g 42 05 42 179 3 7 47 OK Cancel MSODGS22002 185 l astes Hifi Fig 6 23 Mrpl grid with values in the Spot Parameters dialog A grid of Mrpl axes will appear over the Master You can use the grid to verify that the values that you entered make sense you can also use it to estimate the Mrpl values of proteins of interest To see the exact MrpI values of a spot select Spot Parameters from the Spots menu or toolbar and click the spot or use the F6 key command on the spot The MrpI values are displayed in the pop up box Note Ifthe isoelectric point value of the spot falls outside the highest and lowest known values the pI value will not be calculated Empirical results have shown that extrapolated pI values are unreliable due to gel distortions and other factors Transferring Mrpl Data in a Higher Level MatchSet Transfer MrpIs allows you to apply the molecular weight and isoelectric point values from one higher level MatchSet member to the other members This makes it easier to correlate data from the member MatchSets See section 5 8 b Higher Level MatchSets for more information
84. from Gel 3 The Cybergel is a combination of all the spots in which you are interested 9 23 PDQuest User Guide Gels with Narrow pH ranges pH range trom 3 5 pH range trom 4 6 pH range trom 5 7 Cel 1 Gel 2 Gel 2 Cybergel 2 Fig 5 14 Overlapping gels with narrow pH range To combine gels with narrow range pHs select the most acidic gel as your Master template If you select Match gt MatchSet tool you open the Combine Narrow Range Gels or Cybergel dialog 5 24 Matchsets LL Combine Narrow Range Gels E xj To combine narrow range gels D Budd the Master image from the most acidic gel 2 Use this function to add successive gels to the Master The order should correspond to increasing pH The Master will end up being the combination of the gels Add From Member sampled Selection Criteria x Ignore filters for spots outside current Master Range 2058 44 o Max Awg Filter by peak value _ Filter by quality Range 39 o Max z Avg Spots selected inside master az outside master lo x Mark spots to be added Format x Mark add spot location in Master x Auto configure matchset sub windows on launch yf Proceed g Undo add Close Help Fig 5 15 Combine Narrow range gels Use the Add From Member dropdown list to add successive gels to the Master Add additional gels in order of increasing pH The Master or Cybergel becomes a combination of all gel
85. gels to the Master These may be spots induced by your experimental protocols or they may be spots that fall outside the pH gradient of the current Master Auto Adding Spots To automatically add the unmatched spots in a MatchSet member to the Master select Auto Add Spots to Master from the Match menu 5 26 Matchsets i Auto add Spots to Master 3 x Add From Member samp1e3 Selection Criteria PII x 9 Select Member 1 sample3 vl Gaussian 466 spots sampled vl Gaussian 650 spots Cancel x Ignore filters for spots outside current Master _ 522 22 Range 2175 93 Max Avg x Filter by quality al 10 Range 39 Max Avg Spots selected inside master o outside master o x Mark spots to be added o Format x Filter by peak value i Spot Symbol Format E x Style outline 7 filled thick Color yellow blue red green x Auto configure matchset sub windows on launch Symbol ellipse square cross plus d Undo aad Close 2 Help vf Done X Cancel Fig 5 16 Auto add Spots to Master dialog x Mark add spot location in Master In the dialog box select the MatchSet member from the Select Member popup box The MatchSet will reconfigure to display the member and Master in adjacent subwindows with the unmatched spots selected and their corresponding locations in the Master highlighted Selection Cr
86. gives you the option of simply acquiring and displaying a fully exposed image or preserving intermediate exposures Acquire To acquire and display a fully exposed image click on the Acquire button An exposure will be taken based on the time selected in Step III This is appropriate for most short exposures The progress of the exposure will be displayed in the Exposure Status bar at the bottom of the acquisition window Exposure Status A Sr JAcquiring Image Fig G 8 Exposure Status bar when acquiring an image Depending on which dark subtraction type you have selected a dark count may be acquired immediately following image acquisition See Dark Subtraction Type under Options below If you want to stop a scan that is in progress click on the Stop button The acquisition will be terminated Appendix G Fluor S After an image has been acquired a separate window will pop up containing the new image The window will have a default file name that includes the date time and user if known To save the image select Save or Save As from the File menu You can then analyze the image using the analysis functions Live Acquire Live Acquire allows you to view and preserve intermediate exposures leading up to a full exposure This is useful for longer exposure times such as those required by chemiluminescent samples where there is the potential for image saturation When you click on the Live Acquire button the expos
87. if you adjust any of the subsequent controls e g Video Print Image Mode Display controls etc A 5 Step IV Optimize Display The Display controls are useful for quickly adjusting the appearance of your image for output to a video printer Adjusting these controls will automatically freeze the video display and allow you to alter the image within the Gel Doc window Step W Optimize Display High 55 Low E Gamma 1 Highlight Saturated Pixels invert Display ASuto cale Reset Fig A 7 Display controls These controls are similar to those in the Transform dialog box Note The Display controls will only change the appearance of the image They will not change the underlying data High Low Sliders If Auto scale doesn t give you the appearance you want you can use the High and Low sliders to redraw the image yourself In white light mode dragging the High slider handle to the left will make weak signals appear darker In UV mode dragging the High slider handle to the left will make weak signals appear brighter Dragging the Low slider handle to the right will reduce background noise You can also type specific High and Low values in the text boxes next to the sliders Clicking anywhere on the slider bars will move the sliders incrementally Appendix A Gel Doc Gamma Slider Some images may be more effectively visualized if their data are mapped to the computer screen in a nonlinear fashi
88. image f demol vl xl 2D Scan hi Scanset demol MA ES 3 The boundary is created and the spot is added to the Gaussian image demol vl Filtered Modifi demol vl Gaussian Modif dermol vl Raw 2 D Fig 4 14 Creating a freehand boundary To edit the boundary position your cursor on the border and drag across the line with the freehand tool a new white line will appear When you recross the old line a new boundary will be created Deleting and Moving Spot Boundaries To remove a spot boundary you can use the Remove Spot tool or you can click on the spot with the Contour Freehand or Select tool to select it the boundary will change to green and press the Delete key on your keyboard The spot will also be deleted in the Gaussian image 4 21 PDQuest User Guide To reposition a spot boundary make sure the Contour Freehand or Select tool is active then position your cursor in the middle of the spot boundary The cursor will change to a multidirectional arrow simply drag the boundary to a new location The spot will also appear relocated in the Gaussian image Copying Spot Boundaries Between Images To copy a spot boundary between Filtered images first select it the E boundary will change to green then select Copy to Clipboard from the submenu or toolbar Open or select the image you want to copy to and select Paste from Clipboard from the submenu or toolbar The spot boundary will appear in the same rel
89. image in the acquisition window while aligning the two focusing arrows on the camera lens While focusing the camera will limit its focus to a small portion of the sample this will not affect any zoom lens adjustments you may have made When you are finished focusing click on the Stop button G 7 PDQuest User Guide G 4 Step Ill Set Exposure Time When you are ready to capture an image you will need to select an exposure time Exposure refers to the integration of image captures on the CCD over a set period of time The effect is analogous to exposing photographic film to light The exposure time you select should be based on your application and your best guess as to what exposure will give you the best image Note The minimum exposure time in trans illuminated mode is 1 second The minimum exposure time in epi illuminated mode is 0 1 second You can enter an exposure time in seconds directly in the field or use the Arrow buttons to adjust the exposure time in 10 percent increments Step lll Set Exposure Time sec t y Q Previen Fig G 7 Selecting an exposure time The following table provides recommended exposure times for various applications G 8 Appendix G Fluor S Recommended Exposure Times and Lenses Recommended Exposure Lens 4 Filter Accessories Used Fluorescence End Label Gel Colorimetric Gel White Diffusion Plate Colorimetric Blot 0 5 20 sec Zoom IR None X ray film Zoom IR
90. in the Apple Desktop Bus ADB path The ADB port is located on the back of your Macintosh a Fig 1 7 Apple Desktop Bus icon on back of Macintosh PDQuest User Guide The HSK can be inserted at any point in the ADB path between the computer and the keyboard between the keyboard and the mouse between the keyboard and the monitor etc After you have attached the HSK you can restart your computer The code for the Macintosh HSK is QCDIY This code is printed on the key itself 1 7 Starting the Program The Hardware Security Key must be attached to the computer before you can start the software unless you are using a network license Windows The installation program creates an application icon on your desktop To start the program double click on this icon q POQUEST Fig 1 8 Application icon You can also start the program from the Windows Start menu Click on the Start button select Programs select The Discovery Series and select the application name Macintosh After installation the main application folder will be open on your desktop To start the program double click on the application icon shortcut inside the folder You can move this shortcut icon to your desktop Introduction 1 8 Software License When the software opens for the first time you will see a Software License screen that shows the current status of your software license With a new HSK or network license you receive a 30 day te
91. longer exposure times such as those required by chemiluminescent samples where there is the potential for image saturation When you click on the Live Acquire button the exposure time you selected is divided by the number of exposure counts set in the Options dialog box see Options below For example if you enter an Exposure Time of 10 minutes and an Exposure Count of 20 then 20 intermediate exposures will be produced at 30 second intervals Each intermediate exposure will be displayed in the scan window The final full exposure will be displayed in a separate image window Note The first intermediate exposure will take longer than 30 seconds to display if dark subtraction is performed You can automatically save your intermediate exposures as separate files for later review using the Auto Save After Scan option See Options below If you see an intermediate exposure that you like in the scan window click on the Stop button Live Acquire will end and the last intermediate exposure to be completed will open in a separate image window You can then save it for analysis H 6 Options Click on the Options button to open the Options dialog box PDQuest User Guide Fluor 5 Options Fluor S Information Fluor 5 MAs Multilmager simulated Imager Firmware version 1 01 Dark Subtraction Type f Normal f Referenced None Reset Reference Live Exposure Mode Exposure Court E maenas Imager Hardware Version
92. longer to detect spots however spot detection will be more accurate for overlapping spots Note Fewer spots may be found if Gaussian Model During Test is selected because large spots that would be identified as overlapping spot clusters can be correctly resolved with Gaussian modeling 4 1 c Advanced Tab Most of the background removal and smoothing settings under Optional Controls are set automatically based on your selections in Step 1 You should try to Find Spot Centers first before adjusting these controls Note If your image has streaks you should select streak removal under the Streaks tab when finding the spot centers 4 6 Detecting and Editing Spots Streaks Protein gels frequently display streaking in areas of high spot concentration Click the Streaks tab to access the streak removal settings To remove vertical and or horizontal streaks from your image select the appropriate checkbox es The disk radius size is determined automatically based on the size of the largest spot you want to detect Step 1 The default disk radius size is conservative If after you find spot centers you would like to remove more streaking from the image reduce the size of each disk radius by about half and find spot centers again Background Click the Background tab to access the settings for subtracting background intensity from the image Background subtraction is required for spot detection The default method Floating
93. make sure the MatchSet with the duplicates is open and active then go to the Analyze menu open the Replicate Groups submenu and select Create Group 6 9 PDQuest User Guide Replicate Groups Create troup Group Consensus Delete Group Describe Samples b Display Groups Edit Group Normalize Rename Group Fig 6 6 Replicate Groups submenu In the dialog box enter a name for the replicate group then click each duplicate gel in the list to be included in the group The gel names will appear highlighted Click Go to create the group i PDQUEST ES Create Replicate Group Demos 1 xl Member 4gels assigned Select Demos38_1 xl Member 4gel3 assiqned Members Demo39 1 x1 Member 4gels assiqned Demo40_1 xl Member 4gels assigned Fig 6 7 Create Replicate Group dialog box In the upper left status bar in PDQuest you will see the total number of gels in this group Repeat this procedure to designate other groups in the MatchSet To delete a replicate group select Delete Group from the Analyze gt Replicate Groups submenu then select the group to delete from the list To edit a replicate group select Edit Group from the submenu In the dialog add or remove gels from the list To Display Replicate groups you need to specify which groups you want to display from the pop up box To rename a replicate group select Rename Group from the submenu select the group to rename in the dialog then
94. method Click the Add spots to set button Click the mouse on the spots you want to cut When all spots have been added click Save Select menu item Identify Excision Gel selection All the spots will have been selected from the single gel Press Proceed The Integrated Excision Tool will appear Press Align Place the gel on the spot cutter Perform alignment and excision See the PDQuest documentation for further information regarding the previous Steps N 9 Reports N 9 a Report Wizard The WorksBase Custom Report allows you to create reports for various records in the database In the Select Table field select a table from which to pull the records The list of columns from the selected table appears in the Select Columns field Highlight the N 23 PDQuest User Guide columns you want to use in your custom report and click the arrow pointing to the Records to Report field to move the columns to the Records to Report field Use shift click and ctrl click to select multiple columns Once a column is moved to the Records to Report field a hand pointing to the column name appears indicating the column has been moved to the Records to Report field li WorksBase Custom Report P ES Select Table s cSelect Columns Records To Report ADDRESS AGGREGATE_GEL_SPOT AGG_GEL_SPOT_ANNOTATION AGG_GEL_SPOT_MEMBER ANINO_ACID_ANAL_PROTEIN_IDENT ANALYSIS_SEARCH_PTM_TYPE ANNOTATION_CATEGORY ANNOTATION_CAT
95. of an Image To magnify one of nine sections of an image or MatchSet scanset go the 3x3 Sections submenu on the View gt Advanced View submenu and select a region Upper Left Middle Lower Right etc That region will be magnified in the image or MatchSet scanset Location in Gel Location in Gel on the View gt Advanced View submenu displays a graphical overlay on a subwindow or all subwindows showing the region that is magnified green box relative to the entire image blue box Select the command then click the image 3 6 Positioning Images The image positioning tools in PDQuest can be accessed from the main toolbar or View menu or by key command Note that by default if you reposition one image of a MatchSet or scanset in a subwindow all the other subwindows will be repositioned as well To turn off this feature deselect the Auto All Same checkbox under Display Preferences Edit gt Preferences Ctrl F3 will undo your last positioning or other viewing command 3 7 PDQuest User Guide Grab ny Use this tool to drag the image in a window Select Grab from the main toolbar or View menu or position your cursor on the image and press Ctrl G Drag the image then release the mouse button The image will be repositioned Swap The swap command allows you to swap the location of gels in a window This is useful for positioning closely related gels next to one another in a window To use the swap comman
96. on two gels etc The column representing the greatest number of gels lists the number of spots found on all gels This is the column that represents total consensus Ideally only this column should have a non zero value The Ambiguous 6 14 Analysis Tools column lists the number of spots that appear on one gel and can be matched to more than one spot on one or more of the other gels Step 1 Define Acceptance Critena for Presence of Spots Consensus Summary Spots matched across 1 Gel 2 Gels 3 Gels Ambiguous ict Pe First 143 0 second Fig 6 12 Consensus Summary Table Use the Cancel Review and Accept buttons to organize the different Consensus Summary cells according to your review criteria Marking a cell Cancel will color code it red and remove it from automatic inclusion in the list of spots to be reviewed with the Individual Spot Tools later Similarly marking a cell Accept will color code it green and remove it from automatic inclusion in the review list Note that any cell even those marked Cancel or Accept can be manually selected for review later Click Update Consensus to update the Consensus Summary table with any manual changes made in the Review tool Rematch will rematch all the spots in the replicate group Note If you rematch the gels in a replicate group you will lose all prior matching The Global actions and Review tool sections of the Group Review affect the cell you have selected in the Co
97. only need to define two landmarks on your member images to use this command Note Also unlike the regular matching function this command automatically corrects for any rotational errors in the imaged plates common in colony plates You do not need to perform additional image rotation After you have created a MatchSet and defined two landmarks in the member images see above select Colony two point Match from the submenu and click on a member image to automatically match the colonies The other matching commands also work in colony plates Removing Matches To remove all automatic matches from a member image select Remove Auto Matches from the Match gt Edit Matches submenu and click on the member image 9 33 PDQuest User Guide Manual matches and landmarks will not be removed Because this command is irreversible you will be prompted to complete the operation To remove all matches automatic and manual as well as landmarks from a member image select Remove All Matches from the Match gt Edit Matches submenu and click on the member image Because this command is irreversible you will be prompted to complete the operation 5 8 6 Higher Level MatchSets PDQuest allows you to create MatchSets from MatchSets so you can build a database by comparing and summarizing the results of a series of experiments Higher level MatchSets are MatchSets created from the Masters of lower level MatchSets A lower level MatchSet might
98. only require an exposure time of a few seconds which can be quickly adjusted using Manual Expose Manual Expose If you know the approximate exposure time you want you can click on the Manual Expose button Manual Expose is automatically activated after Auto Expose is complete Manual Expose active step Ill Acquire Image button appears selected O Auto y intel Expose Expose Exposure Time sec HE Exposure Time field active adjust number of seconds Fig C 6 Setting a manual exposure With Manual Expose activated you can adjust the exposure time directly by changing the number of seconds in the Exposure Time field Type in a number or use the arrow buttons next to the field When the specified exposure time is reached the last captured image will be displayed in the ChemiDoc XRS image window C 7 PDQuest User Guide Step IIl Acquire Image Auto Manual Live Expose Expose Acquire Exposure Time sec Fig C 7 Freezing the manual exposure Click on Freeze to stop the exposure process Live Acquire Live Acquire mode allows you to specify an interval over which a series of progressively longer exposures are taken All exposures are then displayed on the screen and you can choose the one with the best image Click on the Live Acquire button A settings dialog box will open in which you can specify the total exposure time starting exposure time and numb
99. pixels Mihi GMT lt a did B 11 Ss A o UE a a S B 13 A O dashes nes ASG B 2 C 2 A A T AAN B 5 aTa sre wer ud a RE EEES EAE EA NI B 2 C 2 Video display WINUOW ssssiris ta AT AA B 3 NV TS OPINAS poe des a odas des B 13 Video primine TOOUE INTO si is B 15 Waite ehtmode seee an E a T avant B 5 DE E E E PE EN E A A 6 20 A a E AE 2 15 COlOny amna e anaa a a a a a 5 33 COLOS SEINO erra E R EAEN 3 13 Conme apia a E E E 11 7 OTITIS SOLS ea n N E E A 4 23 Computer Requirements sail saesiiudaa lemtoniedednnsoaan 1 7 Computer requirements O sowneatenancnaneecsedsunmntesuencnuasindestapaoatereoatedemageoshenes 1 8 RR RES 1 7 COMBI UTS WWI WS ai 3 1 Contrast see Transform Crop Savine Crop A e o A aa sateas acanediest 3 28 A aiaobnereSiasbonct se aaiande st ckee Sener inc Gran comenaSoacgoustasiabaauete 3 26 Advanced Crop Command S la dl a 3 28 CUSTOM esas oia oie C 4 Custom OUALION lc clics 3 30 Cybergels Landmark Do solia ica iii esa atan 5 26 Database Node TD asada tected 2 43 Database spot numbers See DSNs Delete Index 4 A Gost ance ald ama neea a 6 10 Denstiy Tol a a 3 11 Densidad oda 3 11 Densi In DO aRo 3 11 PIGUCHOSS SEC 11 eos 3 12 Plot Density Dist bUtiom dd 3 12 Plot Vertical Trace ss prada 3 13 Display setin A o Lo E a le rsonestene EA 2 39 Downloading from Internet O esla 1 16 DSN T ironinen a ES E acces Coase 6 34 A E E a canes 6 35 A a a a a r 6 35 Transferring in high level MatchSets cc
100. possible to compare spots across gels and flag spots of interest manually There are two new commands Save flagged spot set and Clear spot flags Place your cursor on the spot you want to flag and use CNTRL click to flag the spot The cursor becomes an orange circle that shows which spot will be flagged marked for stacking Use Clear spot flags to delete any spots you added Or use SFT CNTRL click to unflag spots If the spot is flagged with a red cross it needs to be added to the Master gel If the spot is flagged with a green cross that means the spot has been matched to the Master gel If images flicker back and forth between the gel members in two windows the flickering cursor becomes a green or red X The Save flag spot set button prompts you to add flagged spots to the Master If you click Yes a pop up box prompts you to name and describe the flagged spot set The flags will then stay in the MatchSet when you close the Image Stack tool To display the spot crosshairs on the images in a MatchSet check the Show Crosshairs box Flags are treated the same as crosshairs If you restart the program reopen the MatchSet the flags are still there 6 6 Analysis Tools Note Spot flags only affect the images you select for display from the list of members 6 3 Scatter Plot Tool The Scatter Plot Tool shows the relatedness of two gels replicate groups or classes in a MatchSet It can also be used to print scatter plots for all the gels
101. recuts or cut previously skipped spots As before click Align to begin the cut run 9 31 PDQuest User Guide a POQUEST Ea Confirm cuts Acquire an image of the MTP in UY mode to check whether re cuts are needed es Mo Fig 9 17 Confirm cuts window Step 1 Answer Yes to bring up the following on screen instructions for imaging the microtiter plate in UV mode fh Image Microtiter Plate Step 1 Setup MTP AN Do not move gel on stage Cover gel with Gel Cutting Sheet Place microtiter plate on Gel Cutting Sheet Close door and click Acquire Image fh Acquire image Step 2 Examine Image Make sure that you can see a visible gel plug in each well used Step 3 Select Action Click on Specify re cuts below to repeat any cuts The Image of the microtiter plate will remain on the screen during re cut selection vf Specify re cuts gt No re cuts Fig 9 18 Image Microtiter plate window Step 2 Do not move the gel or membrane on the cutting platform 9 32 Integrated Excision Tool Step 3 Place a clean plastic Gel Sheet on top of the gel Step 4 Place the microtiter plate on top of the plastic sheet Note the orientation so that you can recognize the A1 location Step 5 Close the Enclosure door Step 6 Click on Acquire image when the plate is in place The UV lights will turn on and the camera will acquire an image with the same exposure time
102. s button N 3 a Filtering You can Filter the list of records by clicking the Filter button The button opens the Explorer Filters dialog You can filter by record name case sensitive User Name and or Date N 10 Appendix N WorksBase ul Explorer Filters a ee x Filter by E Sample Hame contains case sensitive X User name Jetson George f Last week Last month f Range to x Date dd mmm yyyy yf Done x Cancel Fig N 8 Select filter N 4 Image Acquisition Use the acquisition tools as you normally would For example scan a file with the GS 800 When it is done you have the option of saving the image to disk first or simply saving it to the WorksBase database Select Save in WorksBase from the File menu This will bring up the WorksBase file save dialog PDQuest User Guide Ll Save Gel 30degreel 2 in WorksBase as superuser oe E x Image Mame 30degree1 Version 2 Channel o Type Raw 2 D Image Description Scan date 03 Dec 2001 09 35 Scanner Molecular Imager FX Associated records 3 Prooram Customer evaluations a Project Johnson and Johnson Dr Mukherjee ND Experiment a ys Experiment EB S ample iE aoe end Dimen Gel ce Write dataset Dataset for SUPERUSER a Done A Cancel Fig N 9 Save in WorksBase In the Save in WorksBase dialog select or create the Program Project Experiment Sample and Gel records cor
103. s data folder Members Add id sample v1 396 spots o sample v2 260 spots ES Remove sample3 v1 298 spots sampled w1 262 spots tes Master samples v1 305 spots sampleb v1 331 spots Master icon Count E Oj items to reorder them Save X Cancel 2 Help Fig 5 9 Edit MatchSet dialog Note Any annotations analysis sets etc in the MatchSet will be lost if you replace the Master template gel To add a new member to a MatchSet click the Add button Or add a new member gel to the list of Members in the MatchSet using the steps described in Creating a MatchSet 9 13 PDQuest User Guide Removing MatchSet Members To remove a MatchSet member select the gel to be removed from the member list and click the Remove button Since the Remove command cannot be undone a popup box asks you to confirm this action Note Any spots you have added to the MatchSet Master from the removed gel will remain in the Master 5 4 Landmarking Spots Landmarks are required before you can use Classic Matching They are also used to correct errors made by Automated Matching Landmarks are not required for Automated Matching Before you can begin matching spots in the MatchSet you must landmark key spots in all the gels Landmarks are reference spots used to align and position the gels for matching When choosing spots to landmark you should look for spots that are well resolved and present in all membe
104. same area as the analysis image left hand pane 3 Proceed to Step 3 wack Fig 9 11 Position the data area box If the orientation of the image is incorrect use the flip and or rotate buttons to fix the orientation so that it matches the MatchSet image Next position your cursor over the box and drag it so that it defines the same area as the left hand MatchSet image Click OK To reposition the data area box at a later time click the Position Data Box button and drag the box Step 3 Perform Alignment In step three click Auto to have PDQuest automatically align the image If you wish to manually align the image click Manual 9 25 PDQuest User Guide If spots in the cutter image have not aligned correctly with corresponding spots in the analysis image make sure the scanning device has set the correct file size For example the FX Densitometer and the GS series automatically set file sizes whereas camera scanners cannot If your image was generated from a camera acquisition device the Fluor S series the Gel doc ChemiDoc or VersaDoc it will not have specified file dimensions For such images you will need to enter the physical size dimensions correctly at the time of acquisition It is important to have correct physical dimensions at the beginning before you proceed with the spot detection and MatchSet creation otherwise auto alignment will not work correctly Step 4 Alignment Confirmation After the auto ali
105. scan image at which point you can analyze and save it Note The image will open with a default file name that includes the date time and if applicable user name However unless you have selected Auto Save After Scan the file will not be saved until you perform a Save or Save As operation F 13 PDQuest User Guide F 7 Other Options Oversample This feature allows you to scan at the maximum resolution of the scanner and then use spatial averaging to create an image with lower resolution This can result in better images at lower resolution however it takes longer to scan To turn on oversampling click on the Options button in the acquisition window and select the Oversample checkbox in the options dialog With oversampling on you can specify your own resolution within the range of the densitometer by entering values directly in the fields next to X resolution and Y resolution in the main acquisition window Auto Save After Scan To automatically save any scan you create select the Auto Save After Scan checkbox With this checkbox selected when you click on Acquire a Save As dialog box will open asking you to specify a file name and location for the image you are about to create The scan will begin when you click on the Save button Make Backup Copy If you have selected Auto Save After Scan you can also automatically create a backup copy of any scan you create Click on the Make Backup Copy checkbox With this
106. specify the size of your imaging area enter a dimension in the appropriate field When you change one imaging area dimension the other will change to maintain the aspect ratio of the camera lens H 15 PDQuest User Guide The imaging area will change depending on your zoom factor For example if you have zoomed in on a area that is 4 5 x 3 5 cm then you would enter 4 5 for the width 3 5 for the height would be calculated automatically Note Your imaging area settings must be correct if you want to do actual size printing They must also be correct if you want to compare the quantities of objects e g using the Volume Tools in different images The imaging area dimensions also determine the size of the pixels in your image 1 e resolution A smaller imaging area will result in a higher resolution H 6 e Auto scale Transform Auto scale Transform after Acquisition allows the user the option of having the image automatically perform the Auto Scale transform function upon completion of image acquisition This eliminates the need to transform an image or re scan an image when the acquisition time was too short or the iris not opened enough To enable the auto scale transform function check the box labeled Auto Scale Transform after Acquisition in the Options dialog H 7 Other Features Display Options Highlight Saturated Pixels Image file size 2 60 Mb Options Help Fig H 12 Other Fluor S MAX acquisition window feat
107. taken for each of the five possible combinations of camera gain and bin settings These will be retained as the reference dark images until the time is reset again C 13 PDQuest User Guide C 5 d Save Options To automatically create a backup copy of any scan you create select the Make Backup Copy checkbox With this checkbox selected when you save a scan a backup copy will be placed in the same directory as the scanned image Windows backup files will have an sbk extension Macintosh backup files will have the word backup after the file name Note To open a Windows backup file you must first change the file extension To locate the file after changing the extension select All files in the Files of type drop down list of the Open dialog C 5 e Auto scale Transform Auto scale Transform after Acquisition allows the user the option of having the image automatically perform the Auto Scale transform function upon completion of image acquisition This eliminates the need to transform when the acquisition time was too short or the iris not opened enough To enable the auto scale transform function check the box labeled Auto Scale Transform after Acquisition in the Options dialog C 5 f Auto Exposure Threshold When you click on Auto Expose the exposure time is determined by the percentage of saturated pixels you want in your image This field allows you to specify that percentage Typically you will want less t
108. that contain icons for groups of associated functions You can open these toolbars from the main toolbar or from the View gt Toolbars pulldown menu The secondary toolbars can be toggled between vertical horizontal and expanded formats by clicking the resize button on the toolbar itself 2 3 PDQuest User Guide Expanded format Horizontal format Vertical format Seer E Crop a g eizlaaD 30 Rotate 90 Left Vertical Flip a oo E Hl Horizontal Flip Hold cursor over icon to reveal Tool Help Rotate 90 Left Click question marks Rotate 30 Right 7 for on line help Rotate 180 Custom Rotation a Filter Wizard Invert Data 3 gji La ss lee l s e LE Let Click resize button to toggle format SSS Fig 2 3 Secondary toolbar formats and features The expanded toolbar format shows the name of each of the commands Click the Resize button to access the extended format with direct links to the on line Help file for that command 2 1 e Quick Guide The PDQuest Quick Guide accessed from the Help menu is a tool palette that is organized to guide you through the major functions of the software Like the secondary toolbars it can be toggled between vertical horizontal and expanded formats by clicking on the resize button 2 4 General Operation PDQUEST Analysis Quick Guid 2 1 Select Scanner opens Open on line Help Crop Commands are numbere
109. the individual pixels The filter size you select should be larger than the average noise feature but smaller than your data features Note A smaller filter will alter your image less than a larger filter Large filters can result in better suppression of noise but can also blur desirable features in the image 3 35 PDQuest User Guide Step 3 Begin Filtering After you have completed your selections the filter name and size will be displayed at the bottom of the Filter Wizard dialog box To being filtering click the OK button Because filtering is an irreversible process a pop up box will give you the option of filtering the original image creating a copy of the image to filter or cancelling out of this operation If you choose to Copy and Filter you will be asked to enter a new name and or version number for the new copy before the operation is performed Once this information has been entered the filtering operation will be performed 3 15 b Selecting a Filter Directly If you already know the type and size of filter you want you can select it directly by selecting Image gt Filters gt Filter List The pulldown menu includes all the filters available in the Discovery Series The types of filters in the Discovery Series are e Weighted Mean This filter is useful for reducing Gaussian noise it has little effect on salt and pepper It calculates the weighted mean of the pixels within the filtering window and uses it
110. the comparison A pop box will prompt you to select the first member you want to compare Member A Click on the member name A second pop up box will prompt you to select the second member you want to compare Member B When you select the second member the box disappears and the spots in the Master are highlighted a rt Fe a a ad ue EE rin a pat Fig 11 8 A B Overlay example Spots that are marked in both members will be highlighted in green spots that appear only in Member A will appear highlighted in red and spots that appear only in Member B will appear highlighted in blue Spots that appear in neither member will not be highlighted A color key appears at the bottom of the Master Graphs and Reports 11 4 Quantity Table Report If you select Quantity Table Report from the Report menu the Quantity Table Report dialog box opens Ll Quantity Table Report HalfStock 3 E i E x MatchSet name Halftock gt OI Title SS Researcher A ee ee A Institution ee eee a ee eee Which spots Configuration f All matched spots f Individual gels f Analysis set f Replicate groups Optional columns GOSA o Sees _ Mr pl Group E Round to integer _ Trend Lleol e Estimate l Annotation f Exclude Specify 1 o Saturated spots fe Estimate f Exclude Specify a o Sort by fe SSP increasing DSH Trend Rato
111. the eject pin in the cutting tip to eject twice into a microtiter plate well This is useful for membrane blots if the membrane seems to be sticking to the cutting tip No spin when lifting stops the tip from spinning after the cut is made and the tip is lifted from the gel This option may be useful for some gel or blot applications Check the Backed gel setting if you are using gels that have a backing on them The Tip motion range determines the side to side motion in millimeters of the tip as it is cutting backed gels The default range is 2 0 while the maximum range is 5 0 mm The user should determine the optimum setting for their gel Note The backed gel option is recommended for the 1 0 mm cutting tip only Use of the 1 5 mm cutting tip may result in decreased gel pickup efficiency The Gel Defaults button automatically selects the most useful options when cutting gels The PVDF Defaults button automatically selects the most useful options when cutting PVDF membranes 8 2 d Step 4 Set Wash Options Select the wash stations to use after each cut by clicking on one or more checkboxes 8 16 Basic Excision Tool 8 2 8 Performing a Cut Run After you have identified all the cuts you want to make and selected the appropriate options you are ready to begin the cut run If you are using a fluorescent spot cutter close the door of the enclosure before proceeding Click the Begin Resume button You will be prompted to place a mic
112. their total quantity The total number of spots in the image and their total quantity will also be listed i PDQUEST Ea O Low quantity spots Threshold 28 0 oD Spots in Set All Valid Spots Percent 97 spots d spots chal w 1530 0 OD l4332 9 OD 11 Fig 4 15 Pop up box displaying low quantity spot information The low quantity spots will appear highlighted in the image To re highlight the low quantity spots in the set select Spots gt Low Quantity Spots gt Mark Low Quan Spots and click in the image 4 25 PDQuest User Guide To cancel all low quantity spots in the set select Spots gt Low Quantity Spots gt Cancel Low Quan Spots and click in the MatchSet or scanset Gaussian image To restore the cancelled low quantity spots select Spots gt Low Quantity Spots gt Restore Low Quan Spots and click in the MatchSet or scanset Gaussian image Quantity Table You can display the quantity of a spot in each MatchSet gel or replicate group using the Quantity Table function Select Quantity Table from the Spots menu then click on the spot whose quantities in different gels you want to compare Alternatively position the cursor on the spot and click Shift F6 A pop up box will display the quantities of that spot in table format Ll Quantity Table xj SSP 2214 DSH i Group Average LY Court Stock 455 6 1 Halt 151 0 4 3 OR Fig 4 16 Quantity table If Replicate Group Quantitation 1s
113. time the densitometer calibrates an image of the calibration strip will appear in this window F 3 PDQuest User Guide The control panel has been arranged from top to bottom to guide you through the acquisition procedure There are five basic steps to scanning an image using the GS 800 l Ze F 2 Select the application Select the scan area Select the resolution of the scan Calibrate the instrument This is automatic after you enter the step tablet values before you first scan after installation Acquire the image Step I Select Application To set the parameters for your particular scan you can l 2 Select from a list of possible applications or Choose your own filter and light source settings Selecting from the List of Applications When you select from the list of applications the software automatically sets the appropriate filter s and other parameters for that particular application To select from the list of applications click on the Select button under Step I Select Application Appendix F GS 800 ray film Coomassie blue Silver stain Copper stain Zinc stain Stain all Fig F 3 Example of the application tree in the GS 800 dialog box The applications are listed in a tree that expands from left to right First you select the category of your application then you select your particular application To exit the tree without selecting press the Esc key Gray fil
114. to match between gels in red These are also displayed using the Show Symbols F7 command on the Match submenu Choose Cancel Unmatched Spots to eliminate unmatched spots from the MatchSet _ To undo this command select Restore Unmatched Spots Use the Zoom Box tool to study each unmatched spot in detail in each gel If a spot was missed during detection you need to add it to the Gaussian image then match If a spot was not included in the Master you need to add it to the Master If an unmatched spot is not significant you can cancel it Partial Matches Partial matches are spots in the MatchSet Master that are not matched in one or more member images Review these spots to see if they were undetected mismatched cancelled or simply not present Select Mark Partial Matches from the Match gt Edit Matches submenu and click in the Master The partial matches will be circled in the Master 5 31 PDQuest User Guide Graph Partial Matches displays histograms of each partially matched spot in the Master Select the command from the submenu and click in the Master A missing bar in the histogram indicates that the spot is unmatched in a MatchSet gel Missing bars indicate that the spots are not matched in some members SSP 0501 p Fig 5 18 Graph Partial Matches each bar represents the spot s quantity in a gel Note Histograms can only be displayed for one region of an image at a time Use the Reports gt Graph Advance
115. to enter the values for this step tablet you can use the default values in the Step Tablet Values form Attached to the outside of your GS 710 you will find a copy of the manufacturer s printout of the diffuse density values for each segment of your transmissive step tablet as well as a serial number that identifies the step tablet to which those density values belong Those exact density values must be entered into the computer for the software to associate a correct density value with each step on the step tablet E 10 Appendix E GS 710 Note Scanning in transmissive mode with incorrect step tablet values entered into the computer can cause significant errors in the reported densities of your scans To enter the density values for your step tablet click on the Edit Step Tablet button in the Densitometer Options box The Step Tablet Values dialog box for the GS 710 will open Step Tablet Values for GS 710 Transmissive Tablet serial number iosTz64 Quantity Offset 0 00 ll i Step Diffuse Specular Step Diffuse Specular le ollo mio in ao o m o ra co co pa m m m b m i 2 Z0 3 13 3 0 36 O 51 14 2 03 2 87 4 osz foma 15 fis eo 5 0 66 0 93 16 2 34 El p foe mas 17 zas ase 7 fosos meen 18 ss Boe cel 11 i EF 13 2 80 23 936 g 1 27 a 20 4 16 l i H i Ti Pa Pa E ji ALLL ii Cancel Fig E 10 Step Tablet dialog box Help The dialog box wil
116. to increase the amount of signal per pixel However combining the pixels results in a reduction in the resolution of the image G3 Step Il Position Focus Note When you click on the Position or Focus button the light inside the Fluor S box automatically turns on To turn the light off while positioning or focusing hold down the SHIFT key when clicking on the button G 6 Appendix G Fluor S Position After you have selected your application you are ready to center your gel or other object within the camera frame To do so click on the Position button The Fluor S will begin capturing a live image and updating it every second With the Position button selected look at the image in the acquisition window while you position your object in the center of the platen If you have a zoom lens on the camera you can adjust the magnification while you position You can select the Show Alignment Grid checkbox to facilitate positioning Fig G 6 Fluor S alignment grid When you are finished positioning click on the Stop button Focus Note Before focusing you should adjust the f stop on the camera to the lowest setting 1 e the maximum aperture This reduces the depth of field allowing you to more accurately focus the camera Then after focusing increase the f stop to the desired setting See the following table on recommended f stops After you have positioned your sample click on the Focus button and look at the
117. to orksBase on program launch mm Idle time minutes Inter program commands POQUEST port number 5701 WiorksBaze for Proteomics port number 5702 WorksBase Universal Tools port number 5703 yf Done X Cancel E Help Fig N 2 WorksBase Options Connection tab Inter program commands e PDQuest port number e WorksBase for Proteomics port number e WorksBase Universal Tools port number not Available These three are the socket numbers used for communication between the applications They must be set the same in both applications You should only change them if there is a conflict with some other application on your system Save The Save tab of the WorksBase Options dialog box contains options for spot details and excision records Ll WorksBase Options Connection Save Spot details f All spots f All matched spots Annotated amp excised spots f Only excised spots Save gaussian raster image Appendix N WorksBase Login Display Excision records fe Always wite excision records f Never unite excision records f Ask at start of cut run wf Done X Cance 7 Help Fig N 3 WorksBAse Options Save tab Spot details are not required by PDQuest PDQuest saves spot details in large blocks that cannot be read by other programs but are much more efficient If you elect to save spot details here a database record is written for every
118. to perform gel selection and spot detection and do not want to create a MatchSet at this time check the Disable this step box If you do not want to Match Gels at this time you can also check the Disable this step box for Step 4 5 2 Selecting the Master When you first create a MatchSet you need to select one of the images to serve as the template for the MatchSet Master The MatchSet Master is a synthetic image that contains the spot data from all the gels in the MatchSet When you select a gel to serve as the Master template a copy of that Gaussian image is created and designated the Master You then match the spots in the remaining gels with those in the Master and add the unique spots in those gels to the Master Highlight the gel in the member field you have selected to serve as the Master template for the MatchSet Master The Master button is activated and the Master icon appears to the left of the Master gel Select the gel with the greatest number of spots and the least streaking as your Master Choosing the gel with the most spots as the template minimizes the number of spots that you will need to add from the remaining gels e Representative Gel The MatchSet Master is a representation of all the data in your MatchSet and is often used in reports Therefore you may not want to use an atypical gel as a template even if it has the most spots In this case we suggest that you choose the most representative gel in your biologica
119. to replace the value of the pixel being processed e Out of Range Pixel This filter is useful for suppressing salt and pepper noise its effect on Gaussian noise is minimal This filter calculates the mean of the pixel values in the filtering window including the pixel being processed If the difference between the mean and the individual pixel value is above a certain threshold then the individual value is replaced by the mean e Median Also useful for suppressing salt and pepper noise this filter has little effect on Gaussian noise It calculates the median value of the pixels within the filtering window and uses it to replace the value of the pixel being processed The median filter produces very little blurring if a small sized window is selected e Maximum This filter is useful for eliminating pepper noise in an image it worsens the effect of salt noise It replaces the value of the pixel being processed with the maximum value of the pixels within the filtering window e Minimum This filter replaces the value of the pixel being processed with the minimum pixel value within the filtering window This filter is useful for eliminating salt noise in an image it worsens the effect of pepper 3 36 Viewing and Editing Images e MidValue This filter is useful for suppressing uniform noise within an image however it worsens the effect of pepper and salt This filter replaces the value of the pixel being processed with the mean of th
120. use Spot Cutter Settings Click the spot cutter Settings button to open the settings dialog box for the Ye E cutter 9 12 Integrated Excision Tool i Spot Cutter Settings a gt Cutter model Not set O Proteome orke Spot Cutter 25cm e ProteomeW orks Plus Spot Cutter 30cm Microtiter plate well court f 96 wells 304 wells Loading order e Al 42 43 41 861 071 Wash station 1 position a t Test Sige Fark Miscellaneous Cutting tip 1 0mm 1 5 mm diameter Crop left margin a Done X Cancel 2 Help Robot COM 1 COM 2 Fig 9 5 Spot cutter settings dialog box Cutter Model Select the spot cutter model you will be accessing for your cut run This setting most likely will not change unless you have regular access to both types of spot cutters There are slight differences between the two types For instance because the ProteomeWorks Plus spot cutter has a larger platform and cutting tip range it is not necessary to crop the left margin The ProteomeWorks Plus spot cutter also allows you to cut backed gels See Step 2 Select the Cut Run Options on page 9 19 Microtiter Plate Specify the microtiter plate size you are using by clicking on 96 wells or 384 wells 9 13 PDQuest User Guide Specify the order in which you want to the load the wells on the microplate by selecting Al A2 A3 or Al BI Cl This function is useful fo
121. useful when reconciling records between PDQuest and WorksBase for Proteomics Date format This allows you to select the format you would like for date fields N 2 e Login to WorksBase Select Login from the PDQuest gt File gt WorksBase menu to launch the application Your database login prompt displays Ll WorksBase Login x Login Username Madison Password teeters Database JBSLP yf Done x Cancel Fig N 6 Login dialog box Enter your user name and password in the appropriate fields Contact your WorksBase administrator for user name and password information N 8 Appendix N WorksBase If there is more than one database available select the appropriate database by clicking the down arrow If you only have access to one database this field will not appear on the login dialog N 3 Open From WorksBase In the Preferences dialog box if the WorksBase commands are featured is selected Click File gt Open from WorksBase to access the Open from WorksBase dialog If the File based commmands are featured is selected click File gt WorksBase gt Open from WorksBase See Section 2 4 Preferences for further information From this dialog you can open Projects Experiments Experiment Steps Samples 2nd Dim Gels Images and MatchSets When you click one of these options a list of known images MatchSets etc displays in the right window 1 Open from WorksBase as superuser
122. will contain textual annotations the Text option button 7 24 Analysis Sets and Annotations should be selected You can also create specialized categories using the URL arg and Filename buttons category types are described below When you click Done the category is created and will appear selected in the Select Category list To edit an existing category select the category in the list and click the Edit button You can then change the category type and description Note that the annotation entry may be specific to a particular category type changing the type may render the entry meaningless To delete a category select it then click Delete You will be warned before the category and all its entries are deleted URL Categories This type of category provides a link to an Internet protein database or other Web site then the annotation entry references the specific file or Web page for a particular protein You can create different URL categories to link to different databases including Swissprot Unigene and GeneCards To create a URL category open the Add Category dialog box enter a name for the category then select the URL arg option button and type a name for the database or Web page in the Link Name field This is the name that will appear in the Browse Type the entire link address in the Link URL field including http any arguments with a s where the individual spot annotations will go For example Swissprot is
123. you use the GS 710 you must select some calibration settings to ensure that your calibration is accurate E 9 PDQuest User Guide To set the automatic calibration settings click on the More Options button in the GS 710 acquisition window This will open the Densitometer Options dialog box Denzitometer Options On the G5 700 this requires that the calibration overlay be installed Calibrate Before Every Scan Recalibration The Densitometer will be re calibrated Su Interval minutes after this time has expired ope A calibration report will be printed each time the LJ Calibration Report Densitometer is calibrated Calibrate Mow Edit Step Tablet Data Handling The Densitometer will be told to scan at its maximum resolution and spatial averaging will be used to produce the desired resolution This gives better spatial fidelity and lower noise although it takes longer to scan _JOverzample A modified logarithmic scaling system will be used to reduce the size of the data by 50 It introduces a constant relative error of less than 3 TETEN Cancel Help Fig E 9 Densitometer Options dialog box E 5 a Editing the Step Tablet Before you scan for the first time you must enter the step tablet values for your transmissive calibration strip into the Step Tablet Values form Note In reflective scanning the scanner is calibrated against the reflective step tablet However you do not need
124. your computer by a Small Computer System Interface SCSI cable To use the Personal FX you must have a SCSI card installed in your PC If you have a PC with a Windows 98 or Windows ME operating system you may also need to load the SCSI and WinASPI drivers that came with the card Simulation Mode Any of the imaging device acquisition windows can be opened in simulation mode In this mode an acquisition window will open and the controls will appear active but instead of capturing real images the window will create dummy images of manufactured data You do not need to be connected to an imaging device to open a simulated acquisition window This is useful for demonstration purposes or practice scans To enter simulation mode hold down the CTRL key and select the name of the device from the File menu The title of the acquisition window will indicate that it is simulated 1 Personal FX Acquisition Window To acquire images using the Personal FX go to the File menu and select Personal FX The acquisition window for the imager will open displaying a control panel and the scanning area window Appendix I Personal FX pad Personal Fx Step Select Scan Area re Click and drag in diagram to set scan area Top yj Left ae Bottom Ro Right jie C X Quadrant Mode D E Step Il Select Resolution F 50 micrometer G 100 micrometer H
125. 0 o 2 ar Specify re cuts X Nore cuts Step 4 Set O Station 1 x Station Fig 8 17 Microtiter plate after zoom and transform functions applied Step 8 If any of the wells are empty and you want to re cut those spots click on Specify re cuts Then follow these on screen instructions CAES x A Remove Microtiter Plate Return the microtiter plate to the microtiter plate holder Remove Gel Cutting Sheet from the cutting stage Be careful to not move the gell OF Fig 8 18 Message window before recuts are started Step 9 Return the microtiter plate to the plate holder 8 22 Basic Excision Tool Step 10 Remove the plastic sheet from the top of the gel Step 11 Do not move the gel The image and spot selections that were already made still apply to the gel as long as it does not move Step 12 After removing the microtiter plate click OK on the message The Image Microtiter Plate window will close and an image of the microtiter plate will open in front of the Basic Excision Tool screen If you need to resize the window close the recuts box resize the window make any zoom or transform changes using the icons on the top bar above the window then click on the Recuts icon to bring the recuts box back up a 1 MTP Image 1 x1 2D Scan inet 1 PDQUEST Enter wells to be re cut Enter each well as mtp well Separate entries with a semicolon e g 1 401 1 80
126. 0016 SOP 0101 44 11 17 Un narm Un norm Un norm SoP 0102 SoP 0103 SSP 0104 SSP 0105 real 4 Page E of 65 gt mo Reformat Fig 11 12 Quantity Graph Report Viewer The report viewer allows you the option of printing or reformatting the report Click reformat report button to return to the Quantity Graph Report dialog Click on a graph to view the spot in the MatchSet 11 5 Master Image Report Selecting Master Image Report from the Report menu allows you to print a map of up to 300 spots from the MatchSet Master 11 13 PDQuest User Guide In the Master Image dialog you can type in a report title the name of the researcher and the name of the research institution in the appropriate fields Master Image Matchset name ms Report title ms Rezearcher Institution All Matched Specified aan Which spots pele po cade alysis Set Name Go Cancel Fig 11 13 Master Image dialog You can specify the spots that will be included in your report All Matched Spots or the spots in a Specified Analysis Set If you click the Specified Analysis Set button a list of available Analysis Sets displays The name of the set you choose will appear next to the Analysis Set Name prompt Once you have selected your options click Go to open a smaller version of the standard print dialog Select your printer settings and print 11 6 Scatter Plot Report The Scatter Plot Report command on the Report menu allows you to
127. 01 has been missed Cut 1 A02 was made correctly Fig 8 12 Confirmation image note that the first cut made incorrectly When you have confirmed that the cuts are being made in the correct locations click Begin Resume to proceed with the rest of the cuts Otherwise you can abort the run Ending the Cut Run Click Pause to pause the cut run You will be prompted to confirm the pause Note that the cutter will pause only after the most recent cut has been deposited in a well Any remaining cut requests will remain selected in the image At this point you can click Begin Resume to complete the remainder of the cuts or abort the run When all cuts are complete the cutting tip will return to home position and you will receive an alert message on the screen At this point you can visually inspect the cut holes for visible spots or take a UV image of the MTP and confirm the cuts 8 18 Basic Excision Tool 8 2 f Recutting Spots You can specify re cuts by clicking the Recuts button below the image window or when using the UV mode follow the on screen directions for imaging the MTP hl PDQUEST ES x Enter wells to be re cut Enter each well az mtp well Separate entres with a semicolon e g 1 401 1 605 1 006 OK Cancel Fig 8 13 Specify the wells to re cut In the pop up box enter the numbers of your recut requests microplate number well number separated by semicolons Click OK to close t
128. 0LP clear or none light type UV white or none and light source trans epi or neither in the Fluor S MAX for that particular application For applications involving trans illumination you must also specify a scan dimension see below Your selection will be displayed below the Select button To exit the tree without selecting press the ESC key H 4 Appendix H Fluor S MAX Custom Applications If your application is not listed if you want to use a user installed filter or if you want to access Ultra Sensitivity mode see below you can create and save your own custom application From the application tree select Custom then Create This will open a dialog box in which you can name your application and select your settings Mucleic Acid Gels Protein Gels Quantity One Blotting Lreate anew Fluors Application Densitometry Custom AAA Name Gel Type 44x Filter User Filter Illumination White Epi Mode High Sensitivity Cancel OR Fig H 4 Creating a new custom application To select the filter including user defined type of illumination and camera mode click on the appropriate buttons Note Under Illumination there is a listing for a spare UV light source This selects the spare UV bulb in the Fluor S MAX Select this light source 1f your main UV bulb fails Enter a name for your application in the Name field Click on OK to implement your changes After you h
129. 1 PDQuest User Guide i Quantity Graph Report HalfStock Be es x MabtchSet name HaltStack Title Researcher Institution Which spots f All matched spots Trend Analysis set f Quantity wf Done X Cancel 2 Help Fig 11 11 Quantity Graph Report dialog box You can type a report title the name of the researcher and the name of the research institution into the appropriate fields You can specify the spots that will be included in your report All Matched Spots or the spots in a Specified Analysis Set If you click Analysis Set click the drop down arrow to display a list of available Analysis Sets The name of the set you choose appears in the field The quantitation mode for the graphs is determined by the configuration selected in the Configure Graphs dialog See Section 11 1 Configure Graphs for further information on determining the quantitation mode You can sort your spots by SSP number DSN Trend or quantity by clicking the appropriate Sort by button Once you have selected your options click Done to open the report viewer 11 12 Graphs and Reports i Quantity Graph Report HalfStock 17 16 T 55 Un morm Un marm Unri rarin Un norm SSP 0001 S5P 0002 S5P 0003 SSF 0004 257 60 E 29 Un noren Un norm g Un norm SSF OU0G SSP 0007 SSP 0009 164 T0 31 Un norm Un norm Un norm SSP 0010 SoP 0011 SSP 0013 52 45 15 Un norm Un norm Un norm S5P 0014 SSP 0015 SSP
130. 1 01 M Save Usa J Auto Save After Scan J uto scale Transtorm After Acquistion Ok Cancel Fig H 10 Options dialog box H 6 a Dark Subtraction Type All CCD cameras accumulate electrons that produce a signal that is indistinguishable from light This dark count adds to the noise in your images In most cases you will want to subtract this dark count from your images Normal The Normal option button selects the default dark subtraction type In this mode after you acquire an image a dark image of the same exposure length will be taken and this will be subtracted from your image The progress of the dark exposure will be displayed in the Exposure Status bar following the regular image exposure Note In Normal mode a dark image is only acquired the first time you perform a scan with particular application and exposure settings If you perform subsequent scans with the same settings no dark exposure will be taken H 12 Appendix H Fluor S MAX Referenced If you do not want to perform a dark exposure with each acquisition you can take a reference dark exposure that will be saved and subtracted from all subsequent acquisitions Click on the Referenced button to activate this feature The first time you acquire an image after selecting this option the Fluor S MAX will take a 300 second dark exposure that will be saved and used to subtract the dark count from all subsequent a
131. 1 12 31 PM E ETroll 20010907 091 K Auto detect 6 gels 1220 ee ETrol1_20010907_ o ETrol1_20010910_1348 Gel Version sampleb v1 ETrol1_20010910_1525 label Unknown detection Unknown ETroll 20010912 1011 ETroll 20010913 1151 Gel Version sample2 v2 ETrol1_20010913 1312 label Unknown detection Unknown ETroll 20010913 1353 ETroll 0010913 1411 Gel wersion samples v1 ETroll 0010913 1431 label Unknown detection Unknown ETrol1_ 0010913 1443 ETroll 20010921 1500 Gel Yersion sampled wi ETroll 0010974 1107 label Unknown detection Unknown Cancel Gel Version samples w label Unknown detection Unknown Delete Done Fig 2 25 Log report from Spot Detection menu ETrol1_ 20010924 1204 The Micromass Projects path sets the directory where your Micromass project files are stored This path 1s used to link to Micromass project files using the Generate MassLynx Batch File and Import MassLynx Results tools 2 38 General Operation The Micromass Install path sets the directory where your Micromass processing parameters are stored This path is used to link to Micromass parameter files using the Generate MassLynx Batch File function The Temporary path sets the directory where temporary image files and other files generated by PDQuest are stored 2 c Display Preferences 1 Preferences fees x Misc Paths Display Toolbars Application magers Default type Display combined spo
132. 4802 al protein function 5001 hal e 5101 idenmet 5102 mrpi 5103 mut form 5104 te 5105 physy p Ei 5201 plascode 401 pla 5501 m postrmod 5502 tc prot func i 5601 Add Edit Delete Std la O Show description haoz 5803 gt Image Options 11 e 5901 H X Highlight target spot Sot SSP Name Mark all annotated spots Format x Auto select same entry X Mark spots in category Format O Show spot annotations Format 3 Select spots in image J Apply entry Em Close E Help Fig 7 10 Annotation Tool To open the tool with spot s selected select Annotation Select Tool from the Analysis menu and click on a spot in the MatchSet Master or drag a box around a cluster of spots You can also right click on a spot in the Master and select Annotation Tool from the dropdown menu The Annotation Tool will open with the SSP number s of the selected spot s highlighted Alternatively select Annotation Tool from the Analysis menu or toolbar The tool will open with no spots selected Note If you are annotating a MatchSet for the first time you will be prompted to add a list of standard categories when you first open the tool Using the tool you select a category for each annotation select the spot s that you want to annotate in that category and then type the annotation entry Note that the workflow is flexible e g you can select the spot s first then select a categ
133. 5 1 008 O Begin Resume E Setup Display X Well number _ Cut position 293 aa Simulate Xx Cutter X Camera Save Confirm OK Cancel Fig 8 19 Basic Excision Tool fluorescent mode re cuts Step 13 Enter the well numbers of the missed pick up spots in the Re cuts box on the screen Step 14 Click on Begin Resume after the selections are made 8 23 PDQuest User Guide 8 2 9 Saving the Image To save the image taken by the spot cutter click the Save button in the Basic Excision Tool window You will be prompted to enter a name and specify a location for the image before saving 8 3 Other Spot Cutter Controls Click the Move Head buttons under Setup in the Basic Excision Tool e a window to move the cutting tip to the front left corner of the spot cutter or the back right corner You can simulate the action of the cutter and its camera by clicking on the Simulate Cutter and Simulate Camera checkboxes These checkboxes will cut off communication with the cutter and camera 1f selected In simulation mode you will be prompted to open a file image of a gel in place of the camera cutter image 8 24 9 Integrated Excision loo The Integrated Excision Tool allows you to cut spots that have been matched in a MatchSet and placed in an analysis set see section 7 1 Analysis Sets First select the gels from the MatchSet in the Excision Gel Selection EGS Tool that you want to cut f
134. 5 16 Matchsets The number of landmarks required will vary depending on the number and quality of the gels in the MatchSet Gels with well resolved spots and little distortion will require fewer landmarks 5 5 Automated Matching Automated Matching dialog PDQuest includes the ability to match spots with no manual assistance Unlike previous versions you do not have to landmark the images before matching This new facility is now the default method used for matching The previous method is referred to as Classic matching Phases of matching Matching now consists of two phases Primary matching and Extended matching The primary matching phase will make only high confidence matches There is a slider which controls the degree of precision If you specify high precision a spot must be in exactly the expected position based on its neighbors to be matched If you lower the precision the spot is matched even if it is a short distance away from the ideal position You will notice that lowering the precision increases the number of matched spots but may match some spots that should not be matched The more reproducible the gels are the higher the precision setting may be set Primary matches are shown in green Check the Enable overlapped pH range matching box to create Cybergels This will match gels that only partially overlap Since this can slow down the matching process considerably it is not normally set The extended matching
135. 6 Hardware Security Key Hs TO id 1 10 IS A A A 1 12 LS SOL Ware LICE SO di ii 1 13 1 9 Downloading from the Internet ooocccccnnnnnnnnnnnnnnnnnnnonononnnnnnnnnnonnnononnnnnnnos 1 16 LIO POQUest Basicas a occ soacidd 1 17 Ltd Contactins Bio ad ld dls 1 17 General ODerat N ista 2 1 Za Graphical Interface yu is 2 1 2iz WEY DOAL SHONO S id card 2 5 2 52 File Commands and PUNCUIONS searre naie aieeaii 2 7 ZA PARAS and EXPORIN ac 2 20 A 2 21 2 EXPO A DO 2 25 A A O Y 2 35 2 02 INIOUSE aSSICNADIC TOOS sli 2 45 Viewing and Editing Images oncconcccnnccnoncccnanccnancnnanonannnas 3 1 SL Windows and SUBWINGOW Sd 3 1 52 CONMIUrINS SUD W INGOW 8 ua aida acotar 3 1 3 3 Assigning and Interchanging Images oooonoonnnnonnncnonnnnnnnnnnnnnnnnnnnononnnononononnnnonos 3 3 PDQuest User Guide Sd Hine WAT OWS ai ita 3 4 Fy a A O A sueutedisauenntes 3 4 5 03 Positioning Mates ac 3 7 A A sist 3 10 DENSN TOOLS puien an E a E 3 11 O Coapa r sauaMuntiet asain ans bcoc bans bacaantecwommacarebasade gt 3 13 SOs NIAC HANNE NAC WEL sides 3 16 Dis Tine OND WAC W CP sasscchcsuarahannad om bi aaaninasactsnGiasei a 3 18 A OFM eenig enon e ste a ENEE AREO SO 3 20 SIS CTO ppInO MMA Ces ada dc 3 26 3 14 Flipping and Rotating Images ooooccnnnccnncnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnonnnnnnnonnnnos 3 29 Jeli Fene a eS 522565 cag a Gosdens tangs i waamnaagnetensiteses 3 32 De Os MANY CRD AU a ass soate E chante E E E S nsec thas cde tenetac
136. 6 1 SAUS nidos 4 23 SSP AUMOA liato 6 32 O E aeaf 6 32 Transferring in high level MatchSets ccccccseessesseeseeeeeceseeeeeeeeeeeeeeeeeees 6 34 Standard Mage iaa 11 13 Standard Mao nl eiii 5 9 ELEC lI asalariado anecdotas irecoio cagas 5 9 Standard image in a MatchSet AGNES pOl utili 5 26 Standard spot numbers See SSP numbers SANSE Prostate Er E AOE 1 12 Statistical Ama lySIS SEE crar naaa roda 7 12 STATUS DORES ccoo 2 3 SIS mal aaa C 4 Subwindows BENV O O 2 40 CONSI 3 1 B i a eb Live y RPP TOO TEO O emerre arian ene rnin rena yen reren taree tent sateen nner ry 3 1 Technical Service contacts rana 1 17 Text Overlays CEA A aa 3 38 Index 17 PDQuest User Guide U y EGN csn R S E OOl E O E N IOWI DO AA E PE A Three by three 3x3 sections viewing eesseeeeeeeeeseeeees TIFF files A TUE TACOS ati S E A O II N Toolbars O Tools mouse assignable ccccccccnnnncnnnnnnnnnnnnnnnononononononononons Transfer AOSTA sien ooo le eared teas alada NULO SSC AIC io lalo Gamma sde nd alias NE S Highlight Saturated Pixels o oooonoonnnccnnnnnnnnnnnononono nos Histo Si a miis vast acace accte canons ae deeneewonnensedosauarscuiscolan Invest Display sane URL annotations lt td VersaDoc ACUE aan e NA ACQUITING the MATO sais UD 25s Coss oleadas Control PATE slds Index 18 CUSTOM setine o EC A K 5 Di SODIO e aaa Sto C 13 K 14 XPOS Ne td is lite K 8 EXpostte t
137. A reference image of the plate will be acquired and saved on your computer hard drive The reference image will be applied to the sample image to generate a flat field corrected exposure White Light Illumination Flat Fielding When you first select the checkbox and then acquire an image using the white light conversion screen or the white light transilluminator you will be prompted to remove your sample turn on the white light source and collect an exposure of the white light screen transilluminator see the ChemiDoc XRS User Manual A reference image of the screen transilluminator will be acquired and saved on your computer hard drive The reference image will be applied to the sample image to generate a flat field corrected exposure For subsequent UV or white light trans exposures you will be prompted to either use the appropriate saved flat field image or acquire a new one C 10 Appendix C ChemiDoc XRS Quantity One E a E xj Illumination Flat Fielding Do vou want to use the saved reference image for Image Optimization y Yes X No Fig C 10 Using the saved reference image Note Flat fielding is unavailable for White EPI illumination and chemiluminescence applications C 5 Options Click on the Options button to open the Options dialog box Here you can specify certain settings for your ChemiDoc XRS system C 11 PDQuest User Guide ChemiDoc XRS Options E a X Dark Subtraction Informati
138. ANY OTHER PERSON FOR ANY DAMAGES INCLUDING WITHOUT LIMITATION ANY INCIDENTAL OR CONSEQUENTIAL DAMAGES EXPENSES LOST PROFITS LOST SAVINGS OR OTHER DAMAGES ARISING OUT OF THE USE OR INABILITY TO USE SUCH PROGRAM EVEN IF BIO RAD OR AN AUTHORIZED DEALER HAS BEEN ADVISED OF THE POSSIBILITY OF SUCH DAMAGES OR FOR ANY CLAIM BY ANY OTHER PARTY SOME STATES DO NOT ALLOW THE LIMITATION OR EXCLUSION OF INCIDENTAL OR CONSEQUENTIAL DAMAGES FOR CONSUMER PRODUCTS SO THE ABOVE LIMITATIONS OR EXCLUSIONS MAY NOT APPLY TO YOU GENERAL You may not sublicense assign or transfer the license or the Program except as expressly provided in this Agreement Any attempt to otherwise sublicense assign or transfer any of the rights duties or obligations hereunder is null and void and not merely voidable If you have any questions concerning this Agreement including warranty service you should contact BIO RAD LABORATORIES INC 1000 Alfred Nobel Drive Hercules CA 94547 This Agreement shall be governed by the laws of the state of California as applicable to contracts between residents of California which are entered into and performed entirely within California YOU HAVE ACKNOWLEDGED THAT YOU HAVE READ THIS AGREEMENT UNDERSTAND IT AND AGREE TO BE BOUND BY ITS TERMS AND CONDITIONS YOU FURTHER AGREE THAT IT IS THE COMPLETE AND EXCLUSIVE STATEMENT OF THE AGREEMENT BETWEEN US WHICH SUPERCEDES ANY PROPOSAL OR PRIOR AGREEMENT ORAL OR WRITTEN AND ANY OTHER
139. ASE Expression vector pPK113 31509 21 Fig 10 3 Import Mascot results Set Search Filters In this step of the Import Mascot Results dialog select your search filters The currently open MatchSet determines the MatchSet name If you wish to use the MatchSet name check the box to the left of the field To search for a specific cut list or plate select one from the drop down list You can also choose a user name and a run since filter When you enter data into any of these fields the box is automatically checked when you tab out of the field If you no longer want to use one of these filters uncheck the box 10 8 Mass Spectrometry Analysis The Replace Existing data without confirmation check box allows you the option of overwriting current data If this box is unchecked you will receive confirmation every time existing data is about to be overwritten This may be time consuming if you have a considerable amount of data to import which overwrites existing data select Search The Select Search field lists all searches based on the filters selected in step one The search title must be formatted correctly If the search title is incorrectly formatted you will get an error message whether or not you have the search title checked in the options dialog A correctly formatted search title consists of the MatchSet name cut list name cut run serial number plate name MS run sequence number and the SSP number The search title must
140. ATDI O e o a e o a bataenseanetacss 7 15 A aceasta se anal deann tuned A 7 17 Creating annotations from cccccccncnnnnnnnnnnnnnononnnonnnononononnnnnnnnnnnnnnnnnnnnnnnnnnnnaninoss 7 34 CUBIS SPOS TONT ds da 9 2 Ouantta Vestuario gece taiaeecetces 7 8 e A E A 7 12 Analysis LOOSE diana 6 1 ARTO ALON CICE OE S esa a Aae a ea Gene aad 7 24 Pe ENEA ai APA O A O 7 26 POCITAM aida 7 27 UR C atta esc tte cats a casa Canepa E saat tee ttaaeneet 7 25 Amnnotation SELECTOR id 7 23 ANNONO OO li ias 7 23 Eneribs an anota Om is aid 7 28 Maso OPUS cad 7 31 SEE CUA A CAI A OT 7 27 a E A 7 27 Index 1 PDQuest User Guide A E 7 22 A O A a darian nab eematates 7 33 Creatine Tron anal WSIS SETS ala td A A 7 34 IIS DAV ING as 7 31 A A tnt eevrere etre ten 2 33 RIOR Os tea epewateetianlen tenet tombe a ieneou stig aeat a isan sea aaa ea ee eneas 7 28 AA onesies e Pro E O e ania 7 36 Transferring in a high level MatchSet ooocccccccccccnnnnccnnnnonononnnnonnnnnnnnnnnnnos 1 37 AOW CUS oie o orleans 3 8 Assigning images to WINdOWS ccccccccnncnnnnnnnnncnnnonononnnnnonnnnnnnnannnannnnnnnnnnnnnnnnnnnnnnnnnnnss 3 3 AWO ASIME seeno e a n ENE 2 40 Auto add Spots to Standard iaa cios 5 26 Automated detection and Matching in WorksBase ceeeeeeeeeeeeeeeeeeeeeeees N 15 B Basc ACid a a a a a EE 2 43 Batic EX Cision TOOL roekan deals 8 1 ACUE emare aie iaa 8 9 Cut OPTIONS an ad T a E a s 8 16 IASB ODODS 2 AA A E 8 9 Miul
141. Ball and default ball radius size are highly recommended Radius size is calculated automatically based on the largest spot you want to detect selected in Step 1 If Floating Ball results in too much background subtraction or undesirable image artifacts you can select the Roller Ball method Typically the roller ball method results in more image artifacts than the floating ball The Bi Disk method is slightly faster than the floating or roller balls and will perform adequately for images with very smooth even backgrounds However bi disk will perform less well for images with uneven backgrounds smoothing Click the Smoothing tab to access settings for performing smoothing Image smoothing in your scans is optional but highly recommended The default state of the Perform Smoothing checkbox is checked To deactivate this function deselect the checkbox by clicking in it The Filter Type is initially set based on the section of the image you boxed in Step 1 The Kernel Size is set based on the faintest smallest spot you want to detect 4 7 PDQuest User Guide Note You should first attempt to find spot centers using these default settings before attempting to adjust them If you know the type of noise in your images you can select a different filter type and kernel size Section 3 15 Filtering Images provides additional guidance on filter selection e Median This filter is useful for suppressing salt and pepper noise It has l
142. COMMUNICATIONS BETWEEN US RELATING TO THE SUBJECT MATTER OF THIS AGREEMENT
143. E 3 38 DATs EX OV CTIAY S ts 3 38 4 Detecting and Editing Spots coommncccconnnnccncconanonennonannnnnnnas 4 1 4 1 Selecting Spot Detection Parameters cccccesesssseesseeeesseessesessesseeeeeeeeeeees 4 ADs Deren SOUS os 4 9 4 3 Filtered and Gaussian Maes iia 4 12 AA POL Cross Waits and MISES a iaa 4 14 4 5 Adding and Removing Spots cccccccccnnnnnnncncnnnnnnnononnnnnonnononnnonnnnnnnnnnnnnnannnnnno 4 15 AO Spot Boundary OOIS ua iii 4 18 Ae HC AMC CUTS SOUS utilices eden otro eamensbedens 4 22 ALS ODIN SS POS AS 4 23 4 9 Saturated and Faint Spots acia a ea a 4 23 ALLO SPO r QUAMUILY menaa a a a 4 24 Fle SS por QUAY gaian o r a a deaddaawenmadsetaue ne 4 26 AT Ob atan nea adds 4 28 gl Finding SOUS eorn E a e 4 28 Bs Maten SOUS ssion erea A Eai 5 1 Sls Crane a MAES 60 ana a bes havate Wet Uartedes 5 3 Contents ZP electo the Master nd ricas 5 9 Ky VRC MIT SPOS NOAA Op Ao SE LE E TO to cea eta a a lone 5 10 LAO ATA POS A AAA AA canteens 5 14 539 Automated Match coreo ida 5 17 O EDECANES 5 23 gt Te AGING Spots to he Master ntsc dpan 5 26 3 0 Edit Match SU IMC MU dt cdta 5 29 II Matchina Sun dsd 5 37 A nn A 6 1 Ol Spot REVIEW OO ira dio 6 1 6 2 maso Stack Loli odian 6 5 Ad TOON sissies seals a E a a i 6 7 0 JRE plicate Groups saraan a A 6 9 035 Croup CONSENSUS ieee ar E a EA 6 13 60 Sample Database ada iio 6 17 Oe Normalan oN AP OA erm te ee O Se mn ee Cen Re 6 23 05 MiplDaticara
144. EGORY_USE ANNOTATION_USE ANTIBODY_IDENTIFICATION APPLICABLE_RELATIONSHIP_STATES APPLICATION_SPECIFIC_DATA ARRAY POSITION _RESULT ARRAY POSITION TEMPLATE AUTHOR BATCH_INGREDIENTS BINARY_ANNOTATION CELL CELL_ORGANELLE CELL_RELATIONSHIP 3 a AGGREGATE NAME Pr AGGREGATE TYPE EXPERIMENT STEP EXPERIMENT STEP EXPERIMENT STEP EXPERIMENT STEP EXPERIMENT STEP EXPERIMENT STEP EXPERIMENT STEP ar AGGREGATE IDENTIFIER AGG_GEL_ SPOT_NATIVE_IDENT a APPLICATION_SPECIFIC DATA ANNOTATI a APPLICATION_SPECIFIC_DATA APP_DATA a APPLICATION_SPECIFIC DATA APP_ DATA a APPLICATION SPECIFIC _DATA APP DATA a APPLICATION SPECIFIC DATA APP DATA a APPLICATION SPECIFIC DATA APP DATA COMPOSITE X_COORD COMPOSITE Y_COORD EXPERIMENT STEP_IL EXPERIMENT STEP_N2 EXPT_STEP_DESCRIP1 PROTOCOL SOURCE_TY RESULTS_ANNOTATIOK STEP_ORDER_IN_EXP1 STEP_STATUS GEL_SET ANNOTATION 3 ul x El ne AGGREGATE_IDENTIFIER AGGREGATE NAME ACGREGATE_TYPE AGG_GEL_SPOT_NATIVE_IDENT APPLICATION SPECIFIC DATA ANNOTATION APPLICATION SPECIFIC DATA APP DATA DESC APPLICATION SPECIFIC DATA APP DATA IDE APPLICATION SPECIFIC DATA APP_ DATA NAME APPLICATION SPECIFIC_DATA APP DATA OFF APPLICATION_SPECIFIC_DATA APP_DATA TYPI The order of records in the Records to report field determines the layout of the report Record report X Cancel 4 Help Fi
145. Exposure Time field active adjust number of seconds Fig B 5 Setting a manual exposure With Manual Expose activated you can adjust the exposure time directly by changing the number of seconds in the Exposure Time field Type in a number or use the arrow buttons next to the field In UV or White image mode when the specified exposure time is reached the last captured image will be displayed in the ChemiDoc image window The camera will continue to integrate the image on the CCD updating the display whenever the specified number of seconds is reached B 7 PDQuest User Guide Once you are satisfied with the quality of the displayed image click on the Freeze button to stop the exposure process The last full exposure will be displayed in the image window Step IIl Acquire Image Auto Manual Live oO Expose Q Expose Ocio Exposure Time sec Fig B 6 Freezing the manual exposure Click on Freeze to stop the exposure process In Chemi mode Manual Expose will expose an image over the specified exposure time and then stop automatically Note Freeze is automatically activated if you adjust any of the subsequent controls e g Video Print Image Mode Display controls etc Live Acquire Live Acquire mode allows you to specify an interval over which a series of progressively longer exposures are taken All exposures are then displayed on the screen and you can choose the one with the bes
146. Gray file X ray film Blue film Linear Coomassie Blue Silver stain Gel Copper stain Zinc stain Stain all Positive dark bands light background Photograph Negative light bands dark background Coomassie stain Colloidal gold stain Amido black stain Blot AP Substrate BCIP NBT HRP Substrate 4CN HRP Substrate DAB Fig D 4 Applications available in the GS 700 acquisition window Choosing Your Own Settings If you know the filter and light source settings you want or want to experiment with different settings you can choose them yourself Filter Red x Green _ Blue White Light 7 Reflective e Transmissive Fig D 5 GS 700 Custom Application controls D 5 PDQuest User Guide Next to Filter click on either the Red Green Blue or White checkbox or a combination of two of the first three Red Green Green Blue Red Blue Filter Color Wavelength Application Examples 595 750 nm Coomassie G 250 BCIP NBT Fast Green FCF Methylene Blue 400 530 nm Crocein Scarlet Gray Scale 400 750 nm Silver Stains Copper Stains Film Fig D 6 Examples of filter colors and applications 520 570 nm Coomassie R 250 Basic Fuchsin Next to Light select the appropriate light source e Reflective mode is for scanning opaque mediums such as dried gels on filter paper arrays TLC plates and photographs The scanning dimensions in this mode are 21 cm X 35 cm uncalibrated e Transmissi
147. Hardware Security Key HSK which is included in your software package You must attach the Hardware Security Key to your computer before you can run the software Windows Fig 1 5 PC Hardware Security Key Before proceeding please turn off your computer The HSK attaches to the parallel port on the back of your PC If a printer cable is attached to this port turn off the printer and disconnect it After you have attached the HSK you can attach the printer cable to the key itself and restart your computer and printer Introduction Note Some parallel port devices such as zip drives may be incompatible with HSKs Please check with your peripherals vendor The code for the PC hardware security key is EYYCY This is printed on the key itself You will also need to install the system driver that allows the computer to recognize the HSK Note Windows NT and 2000 users must be in the local administrator group to install the HSK driver To install the driver open the Windows Start menu and select Programs gt The Discovery Series Select Install HASP Hardware Security Key driver to begin installation Note Windows 98 users must reboot their computer after installing the HSK Windows NT and 2000 users do not have to reboot Macintosh 1 o ome a SAS m eee Fig 1 6 Macintosh Hardware Security Key Before proceeding please turn off your Macintosh The Macintosh HSK must be inserted
148. Help Fig 2 29 Imagers The Edit gt Preferences gt Imagers tab contains a list of Bio Rad imaging devices supported by the Discovery Series software These are 1 Gel Doc 2 ChemiDoc 3 GS 700 Imaging Densitometer 4 GS 710 Imaging Densitometer 5 GS 800 Imaging Densitometer 6 Fluor S Multilmager 7 Fluor S MAX Multilmager 8 Fluor S MAX2 Multilmager 9 VersaDoc 10 Personal Molecular Imager FX 2 44 General Operation 11 Molecular Imager FX Note If you check one or all of these devices they display in the File menu You need to restart the program in order for your selections to take effect 2 8 Mouse assignable Tools Mouse and Mouse Macro functions are designed for backwards compatibility with previous versions of PDQuest They are not recommended for new users PDQuest has a number of commands that don t perform actions right away but instead assign a function to your mouse e g Zoom Box Density at Cursor Add Spot Select Mouse from the Edit menu to open the Mouse dialog or Edit gt Mouse macro for a pulldown menu ul Mouse Preferences ee xl a Group 1 Group 2 Group 3 El a e El a a El a a a Group 4 ful Number of Mouse Btrs 3 Binz 2 Bine Mouse Configuration File Hame Load Save Reset Delete Done Fig 2 30 Mouse Preferences 2 45 PDQuest User Guide You can choose from up to four groups of mouse functions and select either two or three
149. ING THIS PACKAGE YOU INDICATE YOUR ACCEPTANCE OF SUCH TERMS AND CONDITIONS IF YOU DO NOT AGREE WITH ANY OF THEM YOU SHOULD PROMPTLY RETURN THE PACKAGE UNOPENED YOUR MONEY WILL BE REFUNDED LICENSE Pursuant to the license granted to you by this Agreement you may a use the Program on a single computer b copy the Program into any computer readable or printed form for back up or modification purposes in support of your use of the Program on the single computer certain programs however may include mechanisms to limit or inhibit copying they are marked copy protected c modify the Program and or merge it into another program for your use on the single computer and d transfer the Program and license to another party if the other party agrees to accept the terms and conditions of this Agreement Any portion of the Program merged into another program shall continue to be subject to the terms and conditions of this Agreement If you must transfer the Program you must at the same time either transfer all copies whether in printed or computer readable form to the same part or destroy any copies not transferred including without limitations all modifications and portions of the Program contained or merged into other programs You must also reproduce and include the copyright notice on any copy modification or portion merged into another program YOU MAY NOT USE COPY MODIFY OR TRANSFER THE PROGRAM OR ANY COPY MODIFICATION OR MERGED PO
150. IS a aida 3 10 Password entera Said ad E T arder 1 15 Personal Molecular Imager FX PROQUITING animas ia ia dis I 6 COMMON DANE satan n cir acudo 1 2 Files 2e Ol Me OCN PER CO E a aa a a I 6 OPL ONS te sialisaattoaks eviinasa sli imita 1 7 Saturated pixels highlighting e o ln 1 7 Savio Me Mae aliarse oil E E EE 6 ei ao AAA o A A intents 1 3 SElSC MM ESOO tii se 5 DEISCHMS SCAN AA sclera a E aE I 4 Simulatio mode tesis riera 1 2 ele E GAGEO O PP A on tat laren siete oatene i enseiu aban oe aaeeaty oie 2 43 Plot DIGA aso lied 6 35 AN 6 33 Index 13 PDQuest User Guide Porup Grap PORRA PRO a Baia seadstsadesgadeaeeduasarsaaaseaaase Guaeeeaes 11 6 Preferences Display GOR Un A 2 39 A A E I ET E 2 36 E Me r EE PATE TAE EEE E EEE TIE E I E E EE ET 2 35 A E a E 2 41 Printing PNOUUGIESIZE MAE Sas r aA e aa a ada 2 22 ADOOS asis 7 36 dee TEPORS idas a 2 23 ITA GES NE ecologica oirlo balacera 2 21 VISO IOUS stat iodo idilio idolos 2 24 ProteomeWorks Spot Cutter PAS TC OOM crac statnencesoeatacesishatcn T aun tnceteaent acestaaatss 8 1 Qualita ive Analysis Clear 7 6 Gual OP DOS es anio 4 26 Quantitation Replicate Tops snaa Reon io 6 9 SIT Cle E E E E E E T ia 6 9 Quantitative Analysis SEIS o Load 7 8 GuantiyaGraphs aaa ada ada cabeasbicoud 11 11 OATES DOS E 4 24 Quan y TADE atada la 4 26 anos al eo aaa e E T 2 18 Registration a AP A E ca oe 1 15 DN IMtmel aida is 1 14 REZON TONIE dada 1 13 Index 14
151. MALDI or MS MS button The Required Project Name is based on the MatchSet name Use the MassLynx Project pulldown list to select the project with this name from the appropriate directory Note If you have mapped the path to the Micromass project directory using the Preferences dialog Edit gt Preferences the correct MassLynx project will be automatically selected See Path Preferences 10 3 PDQuest User Guide In Step 2 the mass spec runs that have been performed in this project are listed Click on arun name to select it In Step 3 the samples in the selected run are listed by microtiter plate well and sample date Click on a sample to select it In Step 4 the primary hits for each sample are listed in alphabetical order The number of peptides percent coverage molecular weight and pH are listed for each protein You can display the Joint Probability or MOWSE score for each hit by selecting the appropriate option button at the bottom of the list Select the protein that you feel is the most likely identification and click Apply to import all the information into spot annotations in PDQuest Note The protein you select will be used as the name for the spot All the hits will be included in a special annotation category called Hit Table If you change your mind about the protein name you can change the information directly in the Spot Annotations dialog or reimport the results with a different selection Click Protein P
152. Mode A r i i M i rStep Ill Select Resolution C 50 micrometer i i i 1 100 micrometer o i i 1 Y 200 micrometer ae i T T 800 micrometer Q z 4 E R 1 f Image file size 13 73 Mb S Q Acquire T U Options J Auto Save After Scan Highlight Saturated Pixels A ao e aA Sher e a ne a e ae A ni Make Backup Copy Hide Grid x Wam On Saturated Pixels Info Help Fig J 2 FX acquisition window The default scanning window is marked by grid lines that divide the area into quadrants There is also an outer box and inner box marked by thicker lines The quadrants are numbered 1 16 left to right and lettered A U top to bottom If you prefer a scanning window measured in centimeters deselect the Quadrant Mode checkbox in the control panel by clicking on it To hide the gridlines click on the Hide Grid checkbox under Options The control panel has been arranged from top to bottom to guide you through the acquisition procedure There are four basic steps to scanning an image using the FX 1 Select the application s 2 Select the scan area J 3 PDQuest User Guide 3 Select the resolution 4 Acquire the image J 2 Step l Select Application In Step 1 you select the appropriate filters and other scanning parameters for the type of gel blot plate or other object that you are imaging Each set of parameters you select is called an application The FX now supports multi chan
153. PDQuest User Guide 1 Click Zoom Box button 2 Drag box on image 3 Boxed region is magnified to fill window i Gel 18237 Iof s il Gel 18237 OF x 12237 yi Gel laget Fig 3 4 Zoom Box tool Zoom In Zoom Out Zoom In Alt F2 View menu and toolbar will magnify an image or all the images in a MatchSet or scanset by a defined percentage The percent magnification is specified under Display Preferences Edit gt Preferences Zoom Out Ctrl F2 View menu and toolbar will reduce the magnification by the same percentage View Entire Image If you have magnified part of an image or moved part of an image out of A view this command will restore the original full view Select View Entire Image from the main toolbar or View menu and click the image or position your cursor on the image and press F1 3 6 Viewing and Editing Images Imitate Zoom Imitate Zoom applies the magnification and positioning of a selected window or subwindow to all open windows and subwindows This command only works on comparable images with similar dimensions Note Imitate Zoom is different than the Auto All Same setting under Display Preferences in that is affects all open windows not just the subwindows in a single window Select a window or subwindow you want to imitate then select Imitate Zoom from the Window menu The other windows will change their display to match that of the active window Magnifying Sections
154. RTION IN WHOLE OR IN PART EXCEPT AS EXPRESSLY PROVIDED FOR IN THIS AGREEMENT If you transfer possession or any copy modification or merged portion of the Program to another party your license is automatically terminated TERM The license is effective until terminated You may terminate it at any other time by destroying the Program together with all copies modifications and merged portions in any form It shall also terminate upon conditions set forth elsewhere in this Agreement or if you fail to comply with any term or condition of this Agreement Upon such termination you must destroy the Program together with all copies modifications and merged portions in any form LIMITED WARRANTY AND REMEDIES BIO RAD warrants the media on which the Program is furnished to be free from defects in materials and workmanship under normal use for a period of ninety 90 days from the date of delivery to you as evidenced by a copy of your receipt This warranty is limited to you and is not transferable During the 90 day warranty period BIO RAD shall a replace any media item not meeting the foregoing warranty and which is returned to BIO RAD or an authorized BIO RAD dealer Authorized Dealer with a copy of your receipt or b if BIO RAD of the Authorized Dealer is unable to deliver a replacement media item which is free of defects in materials or workmanship you may terminate this Agreement by returning the Program and you money shall be refunde
155. S or Mascot uses this file for protein fingerprint identifications Next create a sample list using WorksBase Software for Proteomics or the PLGS web page PLGS only The results of a search are written to WorksBase by a plug in module called PLGS_Helper or Mascot Server The WorksBase installation specialist will configure the PLGS_Helper or Mascot Server to connect to WorksBase As results are recorded in WorksBase the corresponding well changes color to green in the WorksBase for Proteomics container view of the MALDI plate or MTP MS MS You must refresh the tree view F5 to see the change N 20 Appendix N WorksBase N 7 d Import Mass Spec results i Import MassSpec Results from WorksBase Y x gt Step 1 Select instrument Step 3 Select sample Matchset Matts WB test A Ol Matts Arbit test 75 11 Feb 2003 Instrument f MALDI MS MS 4 02 Matts Arbit test 63 11 Feb 2003 4 03 Matts Arbit test 23 11 Feb 2003 Step 2 Select run A 04 Matts Arbit test 46 11 Feb 2003 Cut list Matts Arbit test Cut Mass spec run 20030212 124708 Score e Joint probability MOWSE _ Replace existing entries without confirmation 37 Apply all Ed View identifications gt Step 4 Select primary hit Description Peptides Coverage Score Muy pl SYN1_HUMAN 24 69 50 0 0 0 0 0 00 Putative gustatory receptor 63a 18 62 70 0 0 0 0 0 00 Bark agalutinin p
156. SSF 8420 I SSF 5413 aoP 5415 200 Lin norrm OPM I I SSFP 6406 SSP 5326 Options vf Done E Help 41 DPM I l SSP 5315 SSP 6202 S gt P 0304 SSF 7215 Fig 6 2 Spot Review Tool 6 2 Analysis Tools At the top of the review tool window select All Spots to display histograms of all the spots in the MatchSet or Analysis Set to display histograms of only the spots in a specified set If you select the analysis set option a pop up list of available analysis sets will be displayed Select the set you want to display from the list You can reorder the graphs by clicking the Sort By buttons You can sort by Standard Spots SSP number default or Database Spot DSN if defined If you select Trend the graphs are arranged based on a linear regression model of spot presence and quantity in each member If you select Quantity the graphs are arranged starting with spots of greatest quantity to spots of least quantity To scroll through the pages of histograms click First Prev Next or Last with the red arrows above the graphs If you click on a histogram of a spot that spot will be magnified and highlighted in all the MatchSet gels You can then detect and match the spot using the other tools in PDQuest If you click on a histogram the histogram itself will be highlighted by a blue background indicating that it is selected You can select more than one histogram at a time To deselect a histogram clic
157. Selection Mode 9 5 PDQuest User Guide Auto select Select the Auto select option to automatically select the best gels to cut from based on spot quantity and quality When you choose this option the auto selection parameters will become available and the gels will be selected in the table based on these parameters Spot quantity usually varies among the gels in a MatchSet In general you want to cut from gels in which spot quantity is higher to get more sample The Tolerance ratio is used to determine the range of spot quantity that is acceptable when selecting which gel to cut a particular spot from If the quantity of a spot in only one gel falls within the tolerance ratio that gel will be auto selected for the spot cut However if the quantity of a spot in two or more gels falls within the tolerance ratio then the auto select mechanism will consider which gel will best minimize the time and effort required to switch gels on the spot cutter platform For example if you have five gels in a MatchSet with a tolerance ratio of 1 the lowest setting auto selection specifies different cuts in all five gels requiring to you to change gels in the spot cutter five times However by increasing the tolerance ratio slightly auto selection finds that three gels contain spots whose quantities fall within the specified tolerance and it adjusts the cuts so that only those three gels are selected This will save you considerable time and eff
158. Set has been created subsequent changes in the scanset are not automatically transferred to the MatchSet and visa versa Make sure that both the Filtered image and Gaussian image are loaded before adding and removing spots In a MatchSet use Interchange Images F11 main toolbar to toggle between the two images The tools for adding and removing spots are located on the Spots menu and Edit Spots toolbar Adding Spots If a spot in the Filtered image has not been added to the Gaussian image Fs select Add Spot from the Spots menu or Edit Spots toolbar then click the spot in the Filtered image The spot will be Gaussian modeled and added to the Gaussian image Note Itis also possible to add spots on Raw 2 D scans when the Filtered and the Gaussian images are loaded 4 16 Detecting and Editing Spots Problem Spot is undetected in Result Spot is missing in the the filtered image Gaussian image Solution Add spot in the Result Spot added to Gaussian image filtered image Fig 4 10 Adding a spot Note Use Plot Cross section Ctrl t View gt View Density submenu to aid in accurately positioning new spots Note that the spot boundary tools section 4 6 Spot Boundary Tools can be used to add non Gaussian modeled spots to the Gaussian image In a MatchSet if you add a spot to the Master gel template it is automatically added to the Master Removing Spots To remove incorrectly positioned or misidentified s
159. Transform Auto scale Transform after Acquisition allows the user the option of having the image automatically perform the Auto Scale transform function upon completion of image acquisition This eliminates the need to transform an image or re scan an image when the acquisition time was too short or the iris not opened enough To enable the auto scale transform function check the box labeled Auto Scale Transform after Acquisition in the Options dialog K 7 Other Features Display Options Highlight Saturated Pixels Image file size 2 60 Mb Options Help Fig K 10 Other VersaDoc acquisition window features Highlight Saturated Pixels When this box is checked any saturated pixels in the image will appear highlighted in red in the scan window and in the pop up image window To view hide saturated pixels in the pop up image window use the Transform command K 18 Appendix K VersaDoc File Size of Images Image File Size shows the size of the image file you are about to create This size is determined by the resolution of the camera and any binning you perform when capturing the image If you do not have enough computer memory for the specified file size an error message will appear when you attempt to acquire an image Macintosh users can increase the application memory partition See your Macintosh computer documentation for guidance K 19 PDQuest User Guide K 20 Appendix L Calibration and Merging You
160. a Colormap Mame standard Load Delete Color Group Light Overlay Colors A Light Light Purple o Reset Cancel Help Fig 3 8 Colors dialog box selecting a Color Group Within the Colors dialog box click the Color Group button to select the colors of the particular group of objects you want to change e g pop up boxes image colors etc PDQuest User Guide i POQUEST ES A Select Color Group Light Overlay Colors Dark Overlay Colors Matte Colors Popup Colors Fixed Colors Image Colors Fig 3 9 List of Color Groups Click a color group in the list to select it Changing a Color After you have selected the color group to change click the specific color button to change The Color Edit dialog box will open allowing you to adjust the red green blue RGB values of the color you selected i POQGUEST ES Color Edit ma es Cancel Fig 3 10 Color Edit dialog box Drag the sliders or enter a value in the fields The color of the button will change with your adjustments 3 14 Viewing and Editing Images Saving Selecting a Defined Set of Colors After you have changed the colors within color groups you can save these settings for future use on other images The Colormap Name field displays the name of a defined set of colors and color groups There are several standard colormaps or you can create your own To s
161. a name for the microplate and identify the first well you want to load If you specify a starting well other than the default of Al the skipped wells will be noted in the dialog box Click Done when you have completed your selections A green circle will appear on the image indicating the spot to be cut Note that the circle is the size of the actual cutting tip 1 0 mm or 1 5 mm as specified in the spot cutter Settings dialog box Continue clicking on spots in the image with Add Cut selected to make additional cuts The number of cuts will appear in the Cuts Selected field If you specify more cuts than there are wells in the microplate you will be prompted to name a new plate and starting well for subsequent cuts Moving a Cut Circle To move a cut circle make sure that Add Cut is selected then position your cursor inside the cut circle The appearance of the cursor will change to a multidirectional arrow Drag the circle to a new location and release the mouse button 8 13 PDQuest User Guide Multiple Cuts of the Same Spot You can make multiple cuts of the same spot The cuts can be placed in separate microtiter plate wells or in the same well to increase the volume of sample in that well To place multiple cuts of the same spot in different wells make sure the Multiple Cuts Spot checkbox is UNCHECKED then click on several locations inside the spot to create individual cuts of the same spot Drag these circles so that they t
162. a new image in the Spot Detection Wizard The control panel in the Wizard is designed to guide you through the parameter selection process 4 1 a Step 1 Give Manual Guidance In this step you mark features on the gel scan that will be used to automatically select the correct spot detection parameters 1 Click the Faint Spot button then click on the faintest spot in the scan Sk that you want to detect This will set the sensitivity and minimum peak value parameters 2 If the smallest spot you want to detect is smaller than the faintest M spot click the Small Spot button then click on the smallest spot This will set the size scale parameter 3 Click the Large Spot button then draw a box around the largest spot E on the image that you want to detect This sets the radius of the background subtraction rolling ball and the streak removal rolling disk 4 3 PDQuest User Guide Fig 4 3 Image with large small and faint spots defined 4 1 6 Step 2 Test Settings In this step you first see how many spots are detected based on the settings in Step 1 then you adjust those settings until all significant spots are identified Note Before proceeding if there are streaks in your image select the Vertical and or Horizontal streak removal checkboxes under Optional Controls SE Find Spot Centers Click the Find Spot Centers button Noise smoothing background subtraction and if selected streak removal will be performed an
163. aDoc Application X Mame Custom Application 3 Filter S30DF70 llumination Trans uv Gain 0 5 Binning 1x1 Cancel OF Fig K 4 Creating a new custom application Enter a name for your application in the Name field Next click on the buttons next to the Filter Illumination Gain and Binning fields to change these settings Note Note that if you select trans white illumination you will need to place the white light conversion screen on the sample stage A higher Binning setting 2x2 3x3 provides optimal sensitivity for low light applications such as chemiluminescence In this mode the pixels in the camera are binned e g four pixels are combined into one to increase the amount of signal per pixel without increasing noise Note that combining the pixels results in a reduction in the resolution of the image Selecting a higher Gain 4x provides higher sensitivity without reduced resolution however noise will also increase This is useful for faint signals bright spots will saturate Selecting a lower gain 0 5x is useful for brighter images that tend to saturate A gain setting of 1x provides the greatest dynamic range Click OK to implement your changes After you have created an application you can select it from the application tree by selecting Custom and the name you created Appendix K VersaDoc You can edit a custom application by selecting Custom Edit and
164. ach field You can either type a new value directly in each field or for small adjustments use the arrow buttons to adjust the tip by fractions of a millimeter The cutting tip will move after you press Enter or when you click the arrow buttons Note that the arrow buttons are oriented as if you were facing the front of the spot cutter also if you are facing the cutter higher numbers in the first X field will move the cutting tip to the right while higher numbers in the second Y field will move the cutting tip toward the back of the instrument 8 5 PDQuest User Guide When you are satisfied with the position of the cutting tip click Test to lower the tip into the well The tip should be centered inside the well Click Park to return the tip to its home position Wash Station 1 position Calibrate the position of Wash Station 1 on the cutting platform by setting the cursor in one of the Wash station fields and pressing enter Then follow the steps described above for Well Al Once you have set the position of station 1 the cutter robot will automatically calculate the positions of the remaining wash stations Miscellaneous Settings Specify the diameter of the cutting tip by selecting 1 0 mm or 1 5 mm Note that the cut request circles that you make on the image reflect the approximate size of the cutting tip and will change based on the tip size selection If you are using the ProteomeWorks spot cutter you will need to c
165. ages are not exactly the same size you can use the Crop tool to resize them With at least one image open select Multi Channel Viewer from the View menu The topmost open image will be displayed in the viewer window using the Red channel 1 Multi Channel iewer Channels Display Transform Red sampled vl xl Raw 2 D Image x Red channel xX Auto scale image when assigned Green v i iGreen channel Blue Blue channel Done S Print E Help Fig 3 12 Multi Channel Viewer 3 16 Viewing and Editing Images Note The color channel used to display an image in the viewer has no relation to the filter used when capturing the image The red green and blue channels are simply designed to distinguish different images The image name is displayed in the Red name field at the top of the viewer and the Red Channel checkbox will appear selected To add another image make sure the image is open and click the pulldown button next to the Green or Blue name field Select the image name from the pulldown list Add a third image using the same procedure You can reassign the different images to different channels using the pulldown buttons to the right of the name fields Select lt clear gt from the pulldown list to remove an image from that channel of the viewer Viewing Options To remove a particular color channel from the display click in the checkbox associated with that channel t
166. aging Densitometer Fig F 1 GS 800 Imaging Densitometer Before you can begin scanning images with the GS 800 Imaging Densitometer your instrument must be properly installed and connected with the host computer See the hardware manual for installation startup and operating instructions PC Only A Note About SCSI Cards The GS 800 is connected to your computer by a Small Computer System Interface SCSI cable To use the GS 800 you must have a SCSI card installed in your PC If PDQuest User Guide you have a PC with a Windows 98 or Windows ME operating system you may also need to load the SCSI and WinASPI drivers that came with the card Simulation Mode Any of the imaging device acquisition windows can be opened in simulation mode In this mode an acquisition window will open and the controls will appear active but instead of capturing real images the window will create dummy images of manufactured data You do not need to be connected to an imaging device to open a simulated acquisition window This is useful for demonstration purposes or practice scans To enter simulation mode hold down the Ctrl key and select the name of the device from the File menu The title of the GS 800 acquisition window will indicate that it is simulated Note There is no simulated calibration for densitometers F 1 GS 800 Acquisition Window To begin acquiring images go to the File menu and select GS 800 The acquisition wi
167. al size images image reports and video printouts are located on the File gt Print submenu File Edit View Image Spots Open Ciro Save Ctrl s Save 45 Save All Ctrl Revert to Saved Close Alt F4 Close All Unload Ctrl U Gel Doc Chemi Doc 65 500 Personal Fx Frias Export d Print Image Ctrl F Exit Ciro Print Actual Size Image Report Video Print Fig 2 13 File gt Print submenu 2 20 General Operation 2 5 Printing 1 Page Setup opens a dialog box where you can select the size and source of your paper portrait or landscape orientation and set margins for your report 2 Print Image moves you directly to the print menu You can also send an image to the printer using CNTRL P 3 Print Actual Size prints an actual size copy of the image 4 Image Report prints the image and information about its scan history number of pixels data range etc 5 Video Print prints images and reports to a video printer 2 5 a Printing Images Print Image prints a copy of the active image window and any image overlays that are displayed File gt Print gt Print Image opens the Print dialog box with three tabs General Layout and Paper Quality 2 21 PDQuest User Guide General Layout PapersQuality select Printer Add Printer C LSEPOZ on 561 Gi Acrobat PDF Writer CSO LSBPS1 on LSG1 WHF LaserJet 5 5M PostScript on MBG1 Shatus Ready Location
168. all gels and the overall mean coeficient of variation of the replicate groups 9 38 Matchsets Replicate Group Information The Replicate group information lists the replicate groups with the number of members in the replicate group the number of spots matched to all members and the mean coeficient of variation Other Features From the Matching Summary dialog you can Normalize This opens the Normalization dialog allowing you to normalize the data in the Matchset Rematch This opens the Matching dialog allowing you to rematch the gels in the MatchSet View the Scatter plot Opens the Scatter plot viewer 9 39 PDQuest User Guide 5 40 6 Analysis Tools The tools on the Analyze menu are designed to help you identify and compare spots of interest in a MatchSet Analysis sets and annotations are covered in the following chapter Analyze Identify Report W Replicate Groups Create Group Group Consensus Delete Group Enter Sample Attributes Describe Samples Display Groups Copy Sample Database Normalize Edit Group Pair Samples Rename Group Analysis Set Manager Spot Review Image Stack Scatter Plot nnotate Browse annotations More Annot Tools a Annotation Select Tool Find File For Mrpl Standards Enter MrpI Data Create Annotation From Set Spot Numbers Mark MrpI Spots Print Annotations Display Mrpl Data Import Annotatio
169. alstrip These values should correspond to the calstrip segments as they appear from left to right 1 e if the darkest segments are on the left the values will decrease Range of Segment Values and Spot Quantities The range of segment values should be approximately 2 0 to 2 x 10 The minimum value the Calstrip Record will accept is 2 0 A typical maximum is 2 x 10 but this value may vary The range should span four orders of magnitude Typical spot quantities will range from 10 to 5 x 10 depending on the size of the spot An example of segment values DPM cm for an 35S calstrip are Segment 1 2785790 7 14892 0 13 827 0 2 1593760 8 9207 0 14 516 0 3 1002090 9 5791 0 15 315 0 4 67078 0 10 3670 0 16 134 0 5 40895 0 11 2220 0 17 93 0 6 24686 0 12 1396 0 18 44 0 Such a calstrip will produce spot quantitation values that typically range from 2 4 DPM to 502 6 DPM If you are working with Coomassie gels and you want to make a calstrip you should begin with a stock solution and make approximately 10 serial dilutions each of which will be used to make one segment of the calstrip PDQuest User Guide For example you might begin with a 50 ug ml stock solution of BSA and make segments of the calstrip starting at 50 ug and progressing down to 5 0 ng in constant ratio steps In order to enter numerical values in the Calstrip Record that will produce interpretable spot quantitation values convert all the num
170. ame This opens the Analysis Set Template for that analysis set type As you apply criteria for an analysis set the results appear in the gel or gels you specify in the Analysis Set Template This is helpful in that you can analyze data directly from the template without saving the analysis set to the Analysis Set Manager See the following sections for information pertaining to each type of analysis set Editing and Deleting Analysis Sets If you want to edit an analysis set that already exists click on the button below the Edit heading in the ASM and select Edit from the list This opens the Analysis Set template dialog with the current settings for the selected analysis set Make any necessary changes including changing the name if you choose Click Save when you have finished making changes to the analysis set PDQuest User Guide To delete a saved set click on the button below the Edit heading in the ASM and select Delete from the list This permanently removes the analysis set from the list 7 1 c Qualitative Analysis Sets Qualitative analysis sets contain spots that were not detected in one gel but were detected in the other This test would detect a protein that was expressed under experimental conditions but was not expressed in the control sample or visa versa Note We strongly recommend that all the spots in such an analysis set be double checked for accuracy It is possible that a protein might actually be present in bot
171. an be hidden behind images 2 41 PDQuest User Guide Quick Guide Placement and Toolbar Placement determine which side of the screen the quick guide and toolbars will first open The Placement Behavior setting determines whether a quick guide or toolbar will always pop up in the same place and format Always Auto or whether it will pop up in the last location it was moved to and the last format selected Save Prior The Toolbar Orientation option buttons specify whether toolbars will first appear in a vertical horizontal or expanded format when you open PDQuest In the extended version quick access to context sensitive Help displays in the Quick Reference guide Tool Help Delay allows you to specify the amount of time in seconds the cursor must remain over a toolbar icon before the Tool Help appears First time users may want to specify a short delay to learn the names of the toolbar functions while experienced users can specify a longer delay once they are familiar with the icons Persistence determines how long the Tool Help will linger on the screen after you move the cursor off a toolbar icon 2 e Application Preferences ol Preferences oo xj Misc Paths Display Toolbars Application Imager Matchzet Hame Prefix ms Matchset Name Sequence H Joonzz a Auto match after Landmark pH Gradient f Acid Base f Baze Acid wf DE X Cancel 7 Help Fig 2 28 Application preferences 2 42
172. an curve PDQuest uses Gaussian modeling to generate spots that can be precisely identified and quantitated A Gaussian spot is a three dimensional representation of an original scanned spot Gaussian curves are fitted to the scanned spot in the X and Y dimensions and then additional modeling is performed to create the final Gaussian spot Using Gaussian modeling you can accurately quantitate overlapping spots spots in gel streaks and multiple spots in dense clusters 1 5 PDQuest User Guide Matching and Editing After you have detected the spots in a gel or set of gels you are ready to create a MatchSet A MatchSet is composed of the Raw 2 D Filtered and Gaussian images of the gel s in an experiment In a MatchSet the protein spots from the different gels are matched to each other and are included in a synthetic image called a MatchSet standard The standard includes all the information about the spots in the MatchSet As you match the spots in a MatchSet you will correct the results of automatic spot detection by comparing the Gaussian spots with the original spots in the Filtered image Data Analysis PDQuest provides a variety of analytical tools to help you determine which spots are statistically and scientifically meaningful You can normalize the spot quantities in different gels for more accurate comparison You can define replicate groups of duplicate gels You can create groups of spots that are quantitatively qua
173. ant you can use the High and Low sliders to redraw the image yourself In white light mode and chemi mode dragging the High slider handle to the left will make weak signals appear darker In B 10 Appendix B ChemiDoc UV mode dragging the High slider handle to the left will make weak signals appear brighter Dragging the Low slider handle to the right will reduce background noise You can also type specific High and Low values in the text boxes next to the sliders Clicking anywhere on the slider bars will move the sliders incrementally Gamma Slider Some images may be more effectively visualized if their data are mapped to the computer screen in a nonlinear fashion Adjusting the Gamma slider handle changes the light and dark contrast nonlinearly Highlight Saturated Pixels When this box is checked any saturated pixels in the image will appear highlighted in red in the scan window and in the pop up image window To view hide saturated pixels in the pop up image window use the Image gt Transform command Invert Display This checkbox will switch light spots on a dark background to dark spots on a light background and visa versa This will only affect how the image is displayed on the screen not the actual image data Auto scale Clicking on Auto scale will adjust your displayed image automatically The lightest part of the image will be set to the minimum intensity e g white and the darkest will be set to the maximum intens
174. aring Under Member sharing decide whether you want to make copies of the member files or use the originals If you choose Refer to Originals you will save disk space by not making copies of the member image files Note that you may include the same member files in more than one MatchSet If you do edits such as adding or removing spots will affect all MatchSets that include these files On the other hand if you choose Make Copies the files will be copied to this MatchSet s data directory This enhances the possibility that edits made on the 5 6 Matchsets member images will not affect other MatchSets This option uses more space on and requires you to manage disk utilization more carefully Then check whether or not you want to close the original images after the MatchSet has been created After you have selected the MatchSet members and options click Save You will be prompted to select the directory where you want to store your MatchSet by the Save the MatchSet As dialog box When you have specified the correct file name and directory click Save 5 1 c Create a MatchSet from Automated Detection and Matching The second way to create a MatchSet is directly from the Automated Detection and Matching dialog PDQuest User Guide li Automated Detection and Matching on disk Step 1 Select Gels To Analyze samples Add EF Remove T Master sample4 sample2 sample3 sample Co
175. ars Application Imagers Memory Allowance 512 00 Mb Institute Name Bio Rad Laboratories x Enable MassLynx commands _ Maximize application window x Enable Mascot commands mos aia JEnable DOS filename parsing Get name for Network drives _ Removable drives x Enable Unix filename parsing _ Multiple Mouse Assignment Mode Y OK X Cancel 2 Help Fig 2 23 Preferences dialog box Click the appropriate tab to access groups of related preferences After you have selected your preferences click OK to implement them Some of your preferences will only be implemented by exiting and restarting the program A message will notify you if this is necessary 2 a Misc Preferences Click the Misc tab to access the following preferences The Memory Allowance field allows you to specify the amount of virtual memory allocated for the application at start up The default value of 512 00 megabytes is recommended If you receive a warning message when opening the program that the amount of virtual memory is set too high you can enter a smaller value in this field However this should be considered a temporary fix 2 35 PDQuest User Guide Your institution name can be included in reports if you enter it in the Institute Name field Under Windows the Maximize application window checkbox determines whether the main window occupies the entire computer screen when first opened If this is unchecked the menu and status
176. as the first spot cutter image Step 7 Assess the image on the screen for the wells that need to be recut 41 a ee s im Integrated Excision Tool Cut list Member image Analysis set name demo Z v Gel spots to cut fo W Image Microtiter Plate x Step 1 Setup MTP ANDO not move gel on stage Cover gel with Gel Cutting Sheet Place microtiter plate on Gel Cutting Sheet Close door and click Acquire Image fh Acquire image Step 2 Examine Image in each well used Make sure that you can see a visible gel plug Step 3 Select Action Click on Specify re cuts below to repeat any cuts The image of the microtiter plate will remain on the screen during re cut selection vf Specify re cuts X Nore cuts Fig 9 19 Microtiter plate with fluorescent spots in wells Use the Zoom and transform functions on the right side of the Image Microtiter Plate window to get a better view of the microtiter plate wells 9 33 PDQuest User Guide 1 Integrated Excision Tool Cut list Member image Analysis set name demo E v Gel spots to cut o hi Image Microtiter Plate x Step 1 Setup MTP A cS isos ANDO not move gel on stage A e Se 6 6 ad A 9 a ARA AS gel with Gel Cutting Sheet o k o gt 9 Place microtiter plate on Gel Cutting Sheet b gt 4 000000909 O AA e e e 0 000 9 9 9 9 a OOOO 3 PLRO z
177. ase requires that the Oracle 8 1 7 or 9 2 client software be installed on your system This should have been performed by the WorksBase installation engineer The WorksBase encrypted connection string must be installed in the file worksbase cfg which 1s found in the install prm directory of your installation This also should have been done by the installation engineer N 2 Appendix N WorksBase N 2 c Enable WorksBase commands Launch PDQuest and open the preferences dialog found under the Edit Preferences menu On the miscellaneous section of the preferences dialog locate the Enable WorksBase checkbox and make sure it is checked Which commands are featured Below it are two radio buttons File based commands are featured WorkBase commands are featured These commands rearrange the file menu to make the specified set of commands more convenient All commands are always available N 2 d WorksBase options The WorksBase Options dialog box allows you to customize the features of WorksBase in PDQuest Select Edit gt WorksBase Options to open the WorksBase Options dialog box The WorksBase Options dialog box is divided into four separate tabs e Connection e Save e Login e Display Connection The Connections tab of the WorksBase Options dialog box allows you the option of logging in to WorksBase when you open PDQuest PDQuest User Guide i WorksBase Options ON xj Connection Save Login Display Connect
178. aster Note The MatchSets you select must be writable 1 e not locked or read only See your Macintosh or Windows documentation for instructions on making files writable When you have made your selections click the Save button The higher level MatchSet is displayed in a single window with subwindows just like a level 1 MatchSet You can add members to remove members from and reorder members in higher level MatchSets just like level 1 MatchSets These commands are described in 5 1 Creating a MatchSet Landmark and match spots in a higher level MatchSet just as you do in a level 1 MatchSet See 5 4 Landmarking Spots and 5 3 Matching Spots You will be prompted to select a template to use as the Master for the Higher level MatchSet This is similar to selecting the Master template in a level 1 MatchSet Note that when you select the template for the Master you will be prompted to copy the Standard Spot Numbers from that template into the higher level Master Click Yes to copy the numbers or No to assign new numbers You can also transfer numbers from the MatchSet member of your choice later 5 9 Matching Summary The Matching Summary dialog contains information regarding the gels associated with the current MatchSet Open the Matching Summary dialog by selecting Matching Summary from the Match menu 9 37 PDQuest User Guide i Matching Summary for HalfStock Si ot aS x Member informatian Gel Name R
179. atchSet will be listed as well as the number of spots that have been matched between the MatchSets in the higher level MatchSet Next select whether you want to transfer annotations in All Categories or just one category If you select Specified Category select a category from the pulldown list If the two MatchSets share categories selecting Replace Categories will replace each duplicate category and all its entries in the to MatchSet Replace Entries will replace only the entries for shared spots in the to MatchSet If neither checkbox is selected the transfer will only add new entries shared entries and categories will not be replaced Selecting Confirm Before Replacing will prompt you before each category entry is replaced Click Done to complete the transfer An alert box will list how many categories and entries were transferred You can review the transferred annotations using the Annotation Tool 7 38 8 Basic Excision Tool The Basic Excision Tool is used to control Bio Rad s ProteomeWorks spot cutter and ProteomeWorks Plus spot cutter system for basic spot cutting operations If you are cutting spots from MatchSets for mass spec analysis use the Integrated Excision Tool Before using this tool make sure the spot cutter is connected with the computer is switched on and has warmed up and make sure the camera is connected and has been turned on Alternately if you are not connected to a spot cutter you will
180. ate Calibration Report To print out a calibration report each time the densitometer calibrates click on the Calibration Report checkbox D 7 Other Options Oversample This feature allows you to scan at the maximum resolution of the GS 700 42 3 x 42 3 microns and then use spatial averaging to create an image with lower resolution This can result in better images at lower resolution however it takes longer to scan To turn on oversampling click on the More Options button in the acquisition window and click on the Oversample checkbox With oversampling on you can specify your own resolution within the range of 43 169 microns by entering values directly in the fields next to X resolution and Y resolution in the main acquisition window Appendix D GS 700 Auto Save After Scan To automatically save any scan you create click on the Auto Save After Scan checkbox With this checkbox selected when you click on Acquire a Save As dialog box will open asking you to specify a file name and location for the image you are about to create The scan will begin when you click on the Save button Make Backup Copy If you have checked Auto Save After Scan you can also automatically create a backup copy of any scan you create Click on the Make Backup Copy checkbox With this checkbox selected when a scan is created and saved a backup copy will be placed in the same directory as the scanned image Windows backup files will hav
181. ation startup and operating instructions B 1 PDQuest User Guide To use the ChemiDoc you will need to have the Bio Rad supplied acquisition board installed in your PC or Macintosh The drivers for this board will be installed when you install the main software application Make sure that your ChemiDoc camera is turned on If the camera is not turned on the ChemiDoc acquisition window will open and an image capture error will be displayed Simulation Mode Any of the imaging device acquisition windows can be opened in simulation mode In this mode an acquisition window will open and the controls will appear active but instead of capturing real images the window will create dummy images of manufactured data You do not need to be connected to an imaging device to open a simulated acquisition window This is useful for demonstration purposes or practice scans To enter simulation mode hold down the CTRL key and select the name of the device from the File menu The title of the acquisition window will indicate that it is simulated B 1 ChemiDoc Acquisition Window To acquire images using the ChemiDoc go to the File menu and select ChemiDoc The acquisition window for the instrument will open displaying a control panel and a video display window B 2 Appendix B ChemiDoc Chemi Doc Step Position Gel O Live Focus IRIS 200M FOCUS 4 Open Tele Near Y Close Y wide Y Fa C Show A
182. ation you must reboot your computer before using an imaging device Macintosh Insert the Discovery Series CD ROM into your Macintosh The TDS Mac folder will open on your desktop displaying the installers for the Discovery Series applications Double click on the installer for your application Fig 1 4 Installation program icon Macintosh The installer will guide you through a series of screens The installer will create a folder on your hard drive that contains the main application and associated sample images you can select a different folder 1f you wish The installation will also create 1 9 PDQuest User Guide a folder called The Discovery Series in the Preferences folder in your System Folder this contains the Help file and various system files Once installation is complete the folder containing the application icon will appear open on your desktop Before running the software you must install the Hardware Security Key on your computer unless you have installed a computer download version of the software 1 6 Hardware Security Key HSK Note Initial installation of a network server does require the Hardware Security Key included in the software package Installation of an additional Network Client User to a Network License Server System does not require an HSK Please refer to the Network License Installation Guide that ships with Network Licenses Discovery Series software is password protected using a
183. ative position in the new image The boundary will also appear in the corresponding Gaussian image Note that the two images should have the same pixel size and approximate dimensions You may have to reposition the pasted boundary as described above 4 7 Cancelling Spots Cancelling a spot in a MatchSet or scanset preserves its location but does not include it in matching or any other analysis This is different than removing a spot from the Gaussian image entirely Select Cancel from the Spots gt More Edit Tools submenu or toolbar and click on the spot that you want to cancel It will disappear from the Gaussian image it will remain in the Filtered image but will not be included in any analysis To highlight all cancelled spots in the Gaussian image select Cancelled Spots Ctrl F7 from the Spots gt More Edit Tools submenu or Edit Spot toolbar To restore a cancelled spot select Restore from the Spots gt More Edit Tools submenu or toolbar and click on the position of the cancelled spot The spot will reappear To cancel all the spots that have not been matched in a MatchSet select Cancel Unmatched Spots from the Match gt Edit Matches submenu and click in the MatchSet gel 4 22 Detecting and Editing Spots To restore all unmatched cancelled spots in a MatchSet select Restore Unmatched Spots from the Match gt Edit Matches submenu and click in the MatchSet gel 4 8 Combining Spots Combining spots combines the qua
184. available filters However you should experiment with several different filters before selecting the ones that work best for your images The filtering commands are located on the Image menu and toolbar 3 32 Viewing and Editing Images Image Spots Match Analysis Image Tools ES Image Info Ctrl 1 Y cl E w om Dl Go lt 2 y Transform Ctrl B Crop Advanced Crop Vertical Flip Horizontal Flip Rotate Filter Wizard Filter List d Weighted Mean Out OF Range Pixel Invert Data ee Median Reduce File Size Maximum Minimum MidValue PowerMean ContraMean Adaptive Fig 3 21 Filtering commands Note Since filtering is an irreversible process you will be asked if you want to create a copy of the original image before you filter If you are experimenting with various filters you should create copies of your image and compare them side by side If you filter the original image and save it you cannot return to the original unfiltered state 3 15 a Filter Wizard The Filter Wizard is designed to guide you through the filter selection process First you identify the type of noise in your image Next select the size of the filter to use on that noise Finally filter the image 3 33 PDQuest User Guide To open the Wizard select Image gt Filter Wizard or click the Filter Wizard button on the Image Tools toolbar Filter Wizard for Proteins 1D Scan Step
185. ave created an application you can select 1t from the application tree by selecting Custom and the name you created You can delete the application by selecting Custom Delete and the name of the application PDQuest User Guide Scan Dimension If an application uses trans illumination the Scan Dimension buttons become active The scan dimension is the distance traveled by the transilluminating light source as it scans horizontally across the platen Scan Dimension f 80mm 160mm 250mm 300mm Fig H 5 Scan dimension settings for trans illumination The full scanning range is 300 mm Select a smaller range if your sample is small and you do not want to wait while the light source travels over the maximum scan width High Sensitivity and Ultra Sensitivity High Sensitivity and Ultra Sensitivity are different camera modes In Ultra Sensitivity mode the Fluor S MAX camera is cooled to 35 0 degrees C In High Sensitivity mode the camera is cooled to 20 0 degrees C The current camera temperature is displayed at the bottom of the acquisition window Camera Temperature Z 0 Fig H 6 Camera Temperature display High Sensitivity 1s the normal operating mode of the Fluor S MAX Ultra Sensitivity provides optimal sensitivity for low light applications It is the default selection for the chemiluminescence application and may be selected for a custom application Note When you change from High to Ultra Sen
186. ave cut spots from a gel using the Integrated Excision Tool and digested them you can select the parameters for processing them using Micromass s MALDI or MS MS mass spec instruments First you set up a project file using MassLynx software then you export the processing parameters from PDQuest into the project file After you have identified the proteins you can import the results back into PDQuest Note See Micromass s MassLynx documentation for information on project files and how to use them Before you can generate a MassLynx batch file you must map the directories where your Micromass projects and parameters are stored Go the Edit menu select Preferences and click the Paths tab Map the directory where your MassLynx projects are stored using the Micromass Projects path Map the directory where the mass spec parameters are stored using the Micromass Install path See Path Preferences for more information Note These paths must be mapped correctly or you will not be able to access the proper batch file parameters With the MatchSet containing your cut spots open select Generate MassLynx Worksheet from the Identify menu to set up the batch file 10 1 PDQuest User Guide Step 1 In the dialog box the MatchSet name is listed next to MatchSet Select the cut list you want to analyze from the Cut List pulldown list If you have performed more than one cut run on this cut list select the number of the run from the Cut Run
187. be prompted to use a simulated cutter camera Answering Yes to the prompt also opens the Basic Excision dialog Note See the spot cutter hardware manual for instructions on installing the platform camera and associated hardware and software Select Basic Excision Tool from the Identification menu The software will connect with the spot cutter platform and camera and the Basic Excision Tool dialog box will open 8 1 PDQuest User Guide li Basic Excision Tool Step 1 Acquire Image Light on Gel Light off PDF Acquire Image CUY on Fluor Time secs E 000 Step 2 Specify Cuts Pa Add cut Y Remove cut Cuts 0 Excisions 0 Plates 0 Step 3 Set Cutter Options X Spin when cutting _ Cut longer X Spin when ejecting X Double eject No spin when lifting JBacked gel Tip motion range 2 0 Gel defaults PYDF defaults Simulate X Cutter x Camera Fig 8 1 Basic Excision Tool dialog The Basic Excision Tool includes a window for viewing the spot cutter camera image and a control panel for running the spot cutter The Setup buttons are in the lower left hand corner of the dialog 8 1 Spot Cutter Setup Before you can use the spot cutter for the first time you must focus the camera and adjust the cutting tip height You must also adjust the spot cutter settings and calibrate the instrument Basic Excision Tool Setup Calibrate cutter spot cutter setting ae ove head to l
188. ber dialog opens if the images are already open in PDQuest A list of currently open Gaussian images will be displayed Select the images that you want to include in your MatchSet and click Open The images will be included in the Members list of the Create New MatchSet dialog Note In the Select New Member dialog other MatchSets will be displayed as folders from which you can also select Gaussian images the images will be copied from those folder MatchSets into the MatchSet you are creating 5 5 PDQuest User Guide Raw 2 D and Filtered images are in included in the MatchSet by default Including the Filtered images allows you to compare the Gaussian spots with the original spots while matching and edit the results of spot detection Invariably during matching you will notice missed or misdetected spots To view the filtered image click in the Gaussian or Filtered image subwindow and toggle from one version to another Use the Interchange Image F11 command or Interchange All Images Shift F11 Additionally you can place your cursor in the subwindow you want to toggle and right click to display the Quick Reference Guide 1 ee E O view Entre image JUCI Rizo os e Us pa Transtorm iF PEI assign Ye Uh merchange mages wins 7 Spot Detection Wizard Ea Mee la Select Tool o Eg Show Crosshairs l be Hide Overtays P Image Into gnt mage Fig 5 5 Interchange Images 5 1 0 Member Sh
189. bers to ng 1 e multiply all ug values by 10 For the example mentioned above the values would range from 50 000 ng to 5 0 ng saving loading Calstrip Records When you have completed the information in the dialog box click on the Save button at the bottom of the box To access the information in an existing Calstrip Record click on the Load button A list of saved Calstrip Records will be displayed Select the one whose data you wish to see by clicking on it The data associated with that record will be displayed on the form To delete a saved Calstrip Record click on the Delete button Click on the Cancel button to close the Calstrip Record Editor form L 1 b Scan Information After the calibration data has been entered you will also need to enter some information about the gel that goes with it Start by loading the 2 D and calstrip scans Display the calstrip scans by using the Configure Calstrip Windows function Edit gt Calibrate Gels gt Configure Calstrip Windows Click on Gel Record in the Calibrate Gels submenu and click on the calstrip scan The Gel Record dialog box will open in which you can enter information about the calstrip Appendix L Techniques Ll Gel Record Editor 18237 vl x2 0 Scan Gel run date 19 Jul 1989 1 00 Calstrip More User Batch gt Apply Apply To Gel Detection Fluor Autorad Silver Coomassie Copy lees 3H 14c 22 e 355 125 Cancel jaded a
190. blet Values form In the package with your calibration strip you will find a printout of the diffuse density values for each segment of your transmissive calibration strip Those exact density values must be entered into the computer for the software to associate a correct density value with each step on the step tablet Note Scanning in transmissive mode with incorrect step tablet values entered into the form can cause significant errors in the reported densities of your scans To enter the density values for your step tablet click on the Edit Step Tablet button in the Densitometer Options box The Step Tablet Values dialog box for the GS 700 will open PDQuest User Guide Step Tablet Values for GS 700 Transmissive Tablet serial number v10sTz364 Quantity Offset 0 04 p i Step Diffuse Specular Step Diffuse Specular ilies o o Ti o o i pa FR J m m Ha J 2 0 20 0 28 13 1 282 2 66 3 0 36 0 51 14 2 03 2 87 4 fo se om 15 jos Bo 5 0 66 0 93 1h 2 34 a 31 dos pas ees ase 7 osse mee 18 je Boe 2 l 11 LEF 13 2 80 23 96 3 e peo 2 fesa e El ER a ER ER io io Pa pa DJ co co Ha DJ Ti Ti pb i T Pa Pa E 0 l i Cancel Fig D 10 Step Tablet dialog box Help The dialog box will indicate whether the displayed form is for the transmissive or reflective step tablet depending on whether you are in transmissive or reflective mode Note In t
191. boxes will appear across the top of the screen and any toolbars will appear floating on the screen If Enable DOS File Name Parsing is checked for 8 character filenames ending in two digits the final two digits are interpreted as version and exposure numbers For example the file name IMAGE 11 gsc would be parsed as IMAGE ver 1 xpo 1l gsc This is designed to enable backwards compatibility for users with DOS image files You should only check this box if you are using these image files Enable UNIX File Name Parsing is similar to DOS file name parsing Windows and Macintosh users are unlikely to run into difficulties with UNIX parsing therefore this setting is checked by default Multiple Mouse Assignment Mode is designed for backward compatibility with previous versions of PDQuest It is not documented in this manual The Use Custom Open box option to If Get name for Network Drives or Removable Drives are checked the names of these drives will appear in the Custom Open dialog along with the drive letters 2 0 Path Preferences Navigate to the Edit gt Preferences gt Paths tab to set the file paths for various shortcuts and links throughout PDQuest 2 36 General Operation Preferences xl Images C Program Files4Bio Rad The Discovery Senes 5ample Images Browse 2 Default o Matchsets C Program Files Bio RadsT he Discovery SenessSample Matchsets Browse 2 Default Log C Program Files Bic Rad The Discovery Series
192. button to send the Software Registration Form directly to Bio Rad Your information will be submitted and a temporary password will be generated automatically and sent back to your computer Simply continue to run the application as before Bio Rad will confirm your purchase information and generate a permanent license After 2 3 days click on Check License in the Software License screen again to update to a permanent password The Software License screen will not appear automatically after the temporary password has been generated the software will 1 14 Introduction simply open normally Go to the Help menu and select Register to open the Software License screen Registering by Fax or E mail If you do not have Internet access click on the Print button in the Software License Registration Form and fax the form to Bio Rad at the number listed on the form Alternatively you can enter the contents of the form into an e mail and send it to Bio Rad at the address listed in the Registration Form Bio Rad will contact you by fax or e mail in 2 3 days with a full license Entering a Password If you fax or e mail your registration information you will receive a password from Bio Rad You must enter this password manually To enter your password click on Enter Password in the Software License screen If you are not currently in the Software License screen select Register from the Help menu Enter Password ae To registe
193. buttons to assign to each group type the name of the mouse configuration file into the appropriate field Lower button options contain Load Save Reset Delete Done Note Mouse assignable tools selected using the keyboard have slightly different behavior Edit View Image Spots Match Analysis Identification Text Overlays gt Calibrate Gels gt At pointer 0 28 OD Preferences 3x3 average 0 28 OD Ne Colors tousa Example of UE Save Assignments 1 Ctri Shift Mouse assigned Save Assignments 2 Ctrl Shift 2 a cursor Mouse Macro Save Assignments 3 Ctrl Shift rae e Pulldown Menu ursor Save Assignments 4 Ctrl Shift Recall Assignments 1 Ctrl 1 Recall Assignments 2 Ctrl 2 Recall Assignments 3 Ctrl 3 Recall Assignments 4 Ctrl 4 Configure Mouse Fig 2 31 Mouse Macros and Example of a mouse assignable tool When a mouse assignable function is selected the cursor appearance changes The name and icon of the function appear in the status box next to the main toolbar see section 2 1 c The status box displays text according to where your mouse was placed last To deassign a function from the mouse click the toolbar button of the assigned function or click in the status box displaying the assigned function You can also double click the Hide Overlays button 2 46 Note General Operation Some commands in PDQuest become mouse assignable if you are displaying multiple subwindows For examp
194. bwindows Click All to display all available image types in subwindows PDQuest User Guide W Configure Sub Windows for Scanset 18239 EG Loaded 2D Scan Calstrip Filtered Gaussian All images 3 items 3 items 1 item 1 item 8 ho Quick ug FE 1 Gel 18237 Config Windows E Rows x i Images Once or Apply Cancel 18257 v1 Gel Spots 18257 vl xl 2D Scan 18257 vl xl Calstip Son 18257 vl x2 2D Scan 18237 vl x2 Calstrip Soa 18257 vl x3 2D Scan 18237 vl x5 Gakbthp sea 18257 vl Gel image Fig 3 1 Configuring the subwindows of a scanset The Quick Config buttons immediately partition the subwindows into the configuration depicted on the button Note that if you have selected a Loaded Images option only images of that type will be displayed Otherwise select a configuration by typing values in the Windows Rows and Cols fields The Windows field specifies how many subwindows to create Rows and Cols specify how to layer the subwindows For large MatchSets with many members you can deselect the Display Images Once Configured checkbox Then when you apply a new configuration the images will not be immediately refreshed in the windows Click in a subwindow to make the image in that subwindow appear Note Deselect this checkbox only if you have a slow computer and are displaying many images Click the Apply button to implement your configuration selections Viewing and Editing Images
195. c XRS x Step Select Application Select hite Transilluminati White TRANS 2x Gain 1x1 Bin Step Il Position Gel O Live Focus IRIS ZOOM FOCUS 4 Open Tele A Near Y Close Wide Far Step III Acquire Image A Li O rone O Epose Da pe ela X Flat Fielding lo Freeze Display Options Highlight Saturated Pixels Image file size 2 55 Mb Options 7 Help AAA gt Fig C 2 ChemiDoc XRS acquisition window When the ChemiDoc XRS window first opens no image will be displayed The control panel has been arranged from top to bottom to guide you through the acquisition procedure There are three basic steps to acquiring an image using the ChemiDoc XRS 1 Select the application 2 Position and focus the gel or other object to be imaged 3 Acquire the image C 3 PDQuest User Guide C 2 Step Select Application To set the appropriate parameters for the type of object you are imaging click on the Select button under Select Application You have the option of selecting a UV White or Chemiluminescence application You can also select a custom setting Once you select an application the name of the application and its settings appear in the Select application step C 2 a Custom Applications The Bio Rad installed applications have pre set gain and bin settings If the settings for the available applications do not meet your needs you have the option of
196. c feature a motorized zoom lens that can be controlled directly from the acquisition window using the Iris Zoom and Focus arrow buttons Click on the Up Down buttons while viewing your sample in the window to adjust the lens These buttons will not be visible if you are connected to older versions of the ChemiDoc without the motorized zoom lens Step Position Gel 0 Liver Focus IRIS 00M FOCUS Open 4 Tele 4 Near Show Alignment Grid Fig B 3 Live Focus button and camera control buttons You can also select the Show Alignment Grid checkbox to facilitate positioning Note After positioning your sample you should check the Imaging Area dimensions under Options see section B 9 Options to make sure that they conform to the size of the area you are focusing on To determine the size of the area you are B 4 Appendix B ChemiDoc focusing on you can place a ruler in the ChemiDoc cabinet so that it is visible in the image B 3 Step Il Image Mode The Image Mode option buttons change the type and scale of the data as well as the behavior of the ChemiDoc when acquiring an image UV Select this mode for fluorescent samples With this mode selected the data will be measured in linear intensity units White Light Select this mode for reflective and transmissive samples With this mode selected the data will be measured in uncalibrated optical density uOD units Chemi This mode is designed for chemi
197. can checkbox Note In PDQuest this option is preselected and cannot be turned off All images must be automatically saved when acquired With this checkbox selected when you click on Acquire a Save As dialog box will open asking you to specify a file name and location for the image you are about to create The scan will begin when you click on the Save button Make Backup Copy If you have checked Auto Save After Scan you can also automatically create a backup copy of any scan you create Click on the Make Backup Copy checkbox With this checkbox selected when a scan is created and saved a backup copy will be placed in the same directory as the scanned image Windows backup files will have an sbk extension Macintosh backup files will have the word backup after the file name Note To open a Windows backup file you must first change the file extension To locate the file after changing the extension select All files in the Files of type drop down list of the Open dialog E 14 Appendix E GS 710 Highlight Saturated Pixels When this box is checked any saturated pixels in the image will appear highlighted in red in the scan window and in the pop up image window To view hide saturated pixels in the pop up image window use the Image gt Transform command Hide Grid To hide the gridlines in the scanning area window click on the Hide Grid checkbox E 15 PDQuest User Guide E 16 Appendix F GS 800 Im
198. can use calibration strips calstrips to calibrate your scans for more accurate quantitation Calstrips are required for both gel calibration and merging multiple exposures L 1 Calibrating Gels The commands for calibrating gels are located on the Edit gt Calibrate Gels submenu Edit View Image Text Overlays Calibrate Gels Configure Calstip Windows Preferences Edit Calstrip Record Colors Edit Gel Record Hl F ak Box Segment Mouse Macros Remove Segment DefAct vate Segment Plot Calib Curve Display Segment Quan Print Calib Curve Fig L 1 Edit gt Calibrate Gels submenu Creating a Calstrip File If calstrip images are currently part of your gel images you will have to crop them and save them as separate files for use in PDQuest To do so select Advanced Crop gt Define Calstrip Area from the Image menu draw a box around your calstrip then crop it and save it as a calstrip file See section 3 13 a for instructions L 1 PDQuest User Guide Configuring Calstrip Subwindows If you are displaying multiple calstrips linked to multiple exposures see section L 2 Merging Multiple Exposures Configure Calstrip Windows on the Edit gt Calibrate Gels submenu will automatically arrange the calstrip images in subwindows L 1 a Calibration Strip Data Values and units for each segment of your calstrip must be entered into a file called a Calstrip Record prior to calibration To d
199. canner in specular density and therefore must be converted according to the specular diffuse O D ratio This conversion does not affect quantitation gq Specular Light passes directly through medium q Medium gel film etc eS NX lt a Diffuse Light is scattered by medium Fig E 11 Specular and diffuse density Diffuse density values are converted to specular optical density units according to the following formula Specular OD 1 4 Diffuse OD E 12 Appendix E GS 710 E 5 b Calibration Settings After you have entered the step tablet values you can immediately calibrate by clicking on the Calibrate Now button in the Densitometer Options dialog box You can also specify how often you want the GS 710 to automatically recalibrate Either click on the Calibrate Before Every Scan checkbox or enter a Recalibration Interval in minutes in the appropriate field Note The scanner will automatically recalibrate each time you change your filter or your reflective transmissive setting If you select a different application with the same filter and light settings it will not auto recalibrate Calibration Report To print out a calibration report each time the densitometer calibrates click on the Calibration Report checkbox E 6 Acquire the Image Note Before scanning in transmissive mode make sure the white balance region of the scanning area is not covered or obstructed in any way To begin t
200. category When you are finished click Done In the annotation entry for a spot in this category enter the exact file name you want to associate with the spot This file must be located in the directory you specified in the Add Category dialog To open the file linked to a spot right click on the spot in the MatchSet and select Find File For In the pop up box select the appropriate category name This will launch the application associated with that file and open the file You can associate different files with the same spot but they must be linked using different file name categories 7 26 Analysis Sets and Annotations Protein Name Category You can create a special category for identifying spots by their protein names instead of by SSP numbers in the Annotation Tool Open the Add Category dialog box and type protname in the Name field Click Done to create the category In the annotation entry for a spot in this category type the name of the protein for that spot When you select the Sort Name option under the Select Spot s list in the Annotation Tool the spots will be listed alphabetically by their protein names 7 2 Selecting a Category After you have created a category you can select it Click on the category name in the Select Category list Click the Show Description checkbox to display the description of each category in the list All the spots that have been annotated in a selected category will be rr mar
201. ccccsessseseeeeeeeeeceeeeeeeeeeeeeeeeeeeees 6 36 Enter MipEDita anin a a 6 30 Exciston GEN O odos 9 2 A aE E T E E AE 2 20 Exporting ANNON ONS do E a 2 33 ETIE E E E O A EAE E A 2 26 Matehsetdata na 2 28 Exposures VISTE sara aida L 10 PESO S A NS 4 24 File A A a O 2 15 File locations 1 WIndows ta idad 1 9 File transfer between Windows and Macintosh cccccccccnnnnnnnnnnnnnnnnnnnnnnnnononononnnos 2 12 Index 5 PDQuest User Guide PEAY PES ern a Reset Race aceate dn ate eeReahe adap esa eeaethauotasa aka eeaesaiaaeeeteoase a 2 7 Piler W Zad A O O 3 33 Filtered Ima tdi tte 4 12 Prlterins mass NOISE lis 3 32 o A O O O O O N Tr ek eerer etre tee 7 26 iS AAA O O Gonsasiehaons de nies iada ea enaten 4 28 PT PINS MAGES srl ten 3 29 Fluor S MAX Multilmager ACOUIMING CHE TIN ACE castes dad H 10 CoOntobpane le ide ius o iaa als H 3 CUS COMICS E Se oag O E O H 5 Ex poste Me ann O PO Co E O O edaie teas tea mten an H 8 Exposure ies recommended ico H 9 Filesize ot AA A O A H 17 K 19 PP O E NON H 7 ODG ONS aa E E RN H 11 POSI ONIE tl ico H 7 PROVIENE SC I a E E a H 9 Saturated pixels Mie WITS no estoica H 16 SC AMICI SION verns eierens A IA H 6 Selectas an applications ii H 4 sensitivity me hand altra cil cates H 6 ii PP A H 2 Fluor S Multilmager ACONES CAC HMATE saren da G 10 Control panel sunrise cis G 3 CUS COMICS ELIO Surcos rinoceronte aioie ino G 5 Exposure ie load auescaasniee Dead a a dde teas tenet oe G 8 Expos
202. chSet or a spot list exported from some other analysis application Standard Spot SSP numbers are unique numbers that are automatically assigned to each spot in the MatchSet standard when you create the standard Selecting Import list of SSPs from text file option opens the Select file to import dialog Navigate to the directory where the Standard Spot Numbers text file is located As with all other types of analysis sets when the Analysis Set Template is open you can view the results in the MatchSet immediately To the right of the dialog you can specify the spot marker on which gel s to show the spots and what information to show on the gel s When you open the Analysis set Template whatever analysis sets you have selected in the ASM become inactive To view these sets in the Analysis set Template check the box labeled Display Selected sets 7 1 g Boolean Analysis Sets Boolean analysis sets are formed by combining two or more previously defined analysis sets using Boolean operators You can create Boolean sets that include 1 The intersection of two or more sets of spots proteins present in all sets 2 The union of two or more analysis sets all spots in all sets PDQuest User Guide 3 Spots that are unique to set A plus spots that are unique to set B 4 Spots that are found only in set A 5 Spots that are found only in set B Note Boolean analysis of more than two analysis sets is not available for options
203. cified If you select User Specified enter your own scaling factor and units in the appropriate fields 6 7 6 Normalization Methods The normalization method you select will depend in part on how much you know about your gels and how they were prepared In the Normalization dialog box select from the options for your Basis or denominator For example if you can readily identify housekeeping proteins on your gels the Total quantity in analysis set method is preferable If you know that each gel was loaded with a different amount of sample you can select Specified Value and enter the known variation factor Or check the Total quantity in analysis set option and select your set of housekeeping proteins from the pulldown list Total Quantity in Analysis Set This normalization method involves identifying a group of protein spots that are present in all MatchSet members and are conserved 1 e their quantities should not vary between samples These are proteins that you know are expressed in a very consistent manner at all times within a cell with little or no regulation They are commonly referred to as housekeeping proteins Step 1 Create an arbitrary analysis set containing several of these housekeeping proteins from different regions of the MatchSet Master To create an arbitrary analysis set select Analysis Set Manager from the Analyze menu 6 25 PDQuest User Guide Step 2 Click Create then select Arbitrary form the Select S
204. closing File gt Close All closes all open images in all windows _If you have made changes to any of the images a message box will open for each unsaved image giving you the option of saving those changes 2 3 e Saving Files File gt Save will save a new or old scan scanset or MatchSet to your hard disk network drive or other storage media Note In Windows new PDQuest images will be given a gsc extension when they are first saved File gt Save As dialog can be used to save scans scansets and MatchSets under different names and different directories The default name of the file is listed in the Name field along with the type of file for example MatchSet Note that in the case of scansets see section 2 3 a File Types in PDQuest all the different files in the set must be loaded when you select Save As 1f you want the root name to be changed for all of them Save All on the main toolbar or File menu saves all images scansets and or MatchSets that are currently open 2 3 f Revert to Saved File gt Revert to Saved will recover the last saved version of the image or MatchSet you are working on This is a quick way to undo any alterations you have made since you last saved it Any changes you have made since last saving the file will be lost Any open dialog box will also be closed A message box will warn you before completing the command 2 16 General Operation 2 3 9 Image Info On the main toolbar I
205. cquisitions For image exposures of greater or less than 300 seconds the reference dark will be scaled accordingly and then subtracted You can change the default reference dark exposure time using the Reset Reference button see below If you deselect the Referenced button and then reselect it the old reference dark exposure will still be available Note Separate reference dark exposures will be taken for High Sensitivity mode and Ultra Sensitivity mode Once you have created a reference dark in each of these modes each reference dark will be used according to the mode you are in Reset Reference Button If you would like a reference dark with an exposure time that more closely matches that of your typical scans click on the Reset Reference button Reset Reference Dark Image Options Dark Exposure Time 30 oof Seconds OF Cancel Fig H 11 Reset Reference Dark pop up box H 13 PDQuest User Guide A pop up box will prompt you to enter a new reference dark exposure time in seconds Click on OK to implement your change The new reference dark will be created when you acquire your next image Note Because of the high sensitivity of the CCD fluctuations in background radiation and or temperature in the room can affect the level of dark count If you feel that radiation temperature conditions have changed in the room since your last reference dark was created use the Reset Reference button to delete your old refere
206. creating a custom application which allows you to set your own gain and binning settings To use a custom application click Select and choose custom Next select a custom application from the list or select Create to create a new application This opens the custom dialog Create anew ChemiDoc XRS Applications Mame Custom Application 5 Illumination None Gain amp Binning 2x 1x1 Cancel OF Fig 3 3 Creating a custom ChemiDoc XRS application Enter a name for the application From the Illumination drop down list select an 1llumination mode You can select UV white or no illumination Next select gain and binning settings from the drop down list A higher Binning setting 2x2 3x3 provides optimal sensitivity for low light applications such as chemiluminescence In this mode the pixels in the camera are binned e g four pixels are combined into one to increase the amount of signal per pixel without increasing noise Note that combining the pixels results in a reduction in the resolution of the image C 4 Appendix C ChemiDoc XRS Selecting a higher Gain setting 2x provides higher sensitivity without reduced resolution however noise will also increase This is useful for faint signals bright spots will saturate Click OK to save and add the new custom application to the custom list The new application is automatically selected as your application C 3 Step Il Position Gel Cl
207. cts 9 28 Contado CUS ea a a A E Aa 9 30 CUETO UI ONS enee TOTOE OAN 9 19 Exporine Cut iSt aos el E 9 36 NMicrotiter plate Sele CU OM asa a 9 20 Pansin reU TUM cl a a 9 31 RECUS SOUS s E aA iii 9 31 SE Le CUNO CIS anara A 9 2 Selectine eCa SE no r 9 19 A E I A E AE A E T E E A 9 11 Vandan oni AP aE E O a NEOA 9 31 Imerchaneino MMA PCS aerae cae E T A E AEA 3 3 Invert PS 3 38 a A OOA 3 26 Index 10 Index K Keyboard ShO US anaana a a a aE 2 5 L Landmarking Autos match atera aid 2 43 O a essen ee eae eeea aad 5 14 IW APIS dida 5 16 A ANIA ritos 5 16 A o PE OO A N 1 13 A II o AS 1 13 Tire TOON A eae enw erate asset 3 40 Location TM lidad cta 3 10 Lowguality PO dl A aia Reo 4 27 Ia A A A A 4 25 M Macintosh memory assigned to PDQUESE cccccccccnnnnnnnnnnnnnnnnnnnnnnnonnnnnnnononnnanannnnns 1 8 INIA OMIM WING mazes dl da 3 4 Masin Eros PANA yla eo o y E E east 11 6 Mark NMIPESPO A A e ir E euiaehaGses EE 6 30 Partal Mat hes id 5 31 MassLynx Project palis alot lod acia 2 38 Match DEE EE APA RO 5 22 DE A AR A A A E A E T 1 6 Matohi esene ia A sti ensanndee ose waeyeeaetewiaseudoniuiaes 7 20 A 5 17 Index 11 PDQuest User Guide a mere treet Te eer nestor 5 33 Landmann IS 5 14 Bi AA a o O E EC ERE Ren er rire tre Seen e reer enn etver eeretee 5 33 Unimiatehins individual SPOtS os 5 31 Matchset Paria Mate eS a anteaes 5 31 DO tly AUC O OCRE mu EL O 5 22 SE ie N E A A eeeacnes 5 9 Matchsets Adding
208. d The following warranty does not extent to any media item which has been damaged as a result of accident misuse abuse or as a result of service or modification by anyone other than BIO RAD or an Authorized Dealer EXCEPT AS EXPRESSLY SET FORTH ABOVE THE PROGRAM IS PROVIDED AS IS AND NO OTHER WARRANTIES EITHER EXPRESSED OR IMPLIED ARE MADE WITH RESPECT TO THIS PROGRAM INCLUDING WITHOUT LIMITATION THE IMPLIED WARRANTIES OF MECHANTABILITY AND FITNESS FOR A PARTICULAR PURPOSE BIO RAD WITHOUT LIMITATIONS EXPRESSLY DISCLAIMS ALL WARRANTIES NOT STATED HEREIN YOU ASSUME THE ENTIRE RISK AS TO THE QUALITY AND PERFORMANCE OF THE PROGRAM SHOULD THE PROGRAM PROVE DEFECTIVE YOU AND NOT BIO RAD OR AN AUTHORIZED DEALER ASSUME THE ENTIRE COST OF NECESSARY SERVICING REPAIR OR CORRECTION SOME STATES DO NOT ALLOW THE EXCLUSION OF IMPLIED WARRANTIES SO THE ABOVE EXCLUSION MAY NOT APPLY TO YOU THIS WARRANTY GIVES YOU SPECIFIC LEGAL RIGHTS AND YOU MAY ALSO HAVE OTHER RIGHTS WHICH VARY FROM STATE TO STATE BIO RAD DOES NOT WARRANT THAT THE FUNCTIONS CONTAINED IN THE PROGRAM WILL MEET YOUR REQUIREMENTS OR THAT THE OPERATION OF THE PROGRAM WILL BE UNINTERRUPTED OR ERROR FREE YOU ASSUME RESPONSIBILITY FOR THE SELECTION OF THE PROGRAM TO ACHIEVE YOUR INTENDED RESULTS AND FOR THE INSTALLATION USE AND RESULTS OBTAINED FROM THE PROGRAM YOUR SOLE REMEDIES AND BIO RAD S ENTIRE LIABILITY ARE SET FORTH ABOVE IN NO EVENT WILL BIO RAD BE LIABLE TO YOU OR
209. d D SD o a 8 h in step 4 gt oa a Da mi e Spots to be g o matched p E gae hm a a o Slepl rra Sa Step 2 Dota Bow Placement ian k Akyer Lanira rpi pioa Jacque mane ANecoitan Degg coppa image Elimas fe Cut ona E Light on e ae lll E dump ard Contra Conidence at Spot fa muse Lease 7 Light off PVDF Contents Tenia _ as aston OMe bee AA Cul below confidence IM Total cita io Tergat cut eelas i tas man Dro Mara Conestion dese any cul ek on Geena into dia Hi DegP Durg K Ce O He Note Fig 9 13 Matching spots above and below confidence level Note that the green circle represents the actual cut made by the cutting tip in the gel and will vary in size depending on the cutting tip size selected under spot cutter Settings The following display options are available in the Align Analysis and Cutter Images dialog box Cut Marks will mark the spots to be cut with red Xs in the analysis image Matches will mark matched spots with green circles in both the MatchSet image and cutter camera image Auto zoom will automatically change the magnification and position of the right hand camera cutter image to imitate the display in the analysis image 9 27 PDQuest User Guide Cut Area displays a green box on the cutter camera image this shows the boundary of the cutting area The cutting head cannot cut spots outside this area Multiple Cuts d
210. d select Grab from the main toolbar or View menu or position your cursor on the image and press shift G Drag the image to another image then release the mouse button The two images change places Arrow Keys You can shift the displayed region in a subwindow or all subwindows by using the Arrow keys on your keyboard With a window or subwindow selected click an arrow button The image will shift in the direction of the arrow click The amount the image shifts is determined by the Pan setting in Display Preferences under Edit gt Preferences 3 8 Viewing and Editing Images 3 6 a Advanced View Toolbars gt View Entire Image Fl Zoom Box F2 Grab Ctrl 6 Zoom In Alt F2 Zoom Out Ctrl F2 View Density Hide Overlays F4 Advanced view At Cursor F3 Undo Zoom Mowe Ctrl F3 3x3 Sections Upper Left Multi channel Viewer Pan Upper Middle Refresh Upper Right Location in Gel Middle Left All Windows Middle All At Cursor AIL AFS Middle Right All Undo Zoom Move Lower Left 5 iz Same ShiFt F3 Panteft Left Lower Middle Hide All Overlays Shift F4 Lower Right Pan Right Right All 3x3 Sections b PanUp Up Al Pan f Fan Down Down Refresh all nn All Entire Images ShiFt F1 Fig 3 5 Advanced View submenus Centering an Image You can center an image or images in a MatchSet scanset quickly and easily using the At Cursor command F3 on the keyboard also on the View gt Advanced View submenu This
211. d to indicate sequence for scanning the gel analyzing the image and outputting data Spot Detection Wizard Edit Spot Tools cn Match Tools Normalize AEB 88 1 28 eS Let io oo a mp Mrpl Tools 2 3 H l L la lo le la de lr ls Analysis Annotation Tools a Annotation Toal El ER Ei a 10 Graph Tools Resize button S la lo E Fig 2 4 PDQuest Quick Guide In the expanded format the Quick Guide commands are numbered as well as named so that the order of operation 1s clear Simply follow the steps and the Quick Guide will lead you through the analysis Note that many of the buttons open toolbars of related commands e g Match Tools As with the secondary toolbars you can click the next to the name of a function to display Help 2 2 Keyboard Shortcuts Many of the functions may be executed using keyboard commands e g pressing the Fl key will activate the View Entire Image command A list of key combinations and their associated commands will be displayed if you select Keyboard Layout from the Help menu 2 5 PDQuest User Guide Keyboard Layout Unmodified Pan Up Pan Down View Entire Image com Box At Cursor Hide Overlays show Crosshairs Spot Parameters show Symbols Mark Landmarks show Match Pop up Graph Interchange Images Configure Mouse Alternate Configure Sub Windows com In oK All Pan Left 411 Pan Up 411 Pan Ri
212. d Spot Ellipses is checked marked spots e g spots marked using the Saturated Spots command will be displayed as filled ellipses If unchecked they will be displayed as unfilled ellipses 2 40 General Operation Solid Label Background will automatically display spot labels in a filled background box If Small Window Labels is checked any text in the windows subwindows will be displayed in a smaller font 2 d Toolbar Preferences il Preferences ee xj Misc Paths Display Toolbars Application Imagerz O Show Quick Guide at startup Alon Quick Guide with document x Guides always on top Quick Guide placement amp Left Right Toolbar Placement e Left Right Placement behavior e Always auto 7 Save prior Toolbar onentation f Horizontal Vertical Guide expanded Tool help delay secs 2 5 Persistence 3 0 y OK X Cancel 7 Help Fig 2 27 Toolbar preferences Click the Toolbar tab to set the behavior and positioning of the secondary toolbars and Quick Guide If the Show Quick Guide checkbox is selected the quick guide will open automatically when you open PDQuest If Align Quick Guide with Document is selected the quick guide will pop up flush with the edge of your documents Otherwise it will appear flush with the edge of the screen The Guides always on top checkbox determines whether the quick guide and toolbars will always be displayed on top of images or whether they c
213. d gels Unmatched spots in the member gels are marked with red circles Auto matching should successfully match most of the spots in the MatchSet Ch Matchset MSO0002 7001 10 04 Modified Match ale ETE 62 uunatched 2731 po Match symbols Panatened 335 wunatched 07 MS00092 GOTO Masa Samples vh xF Raw 2 D Image P a a 5 y m m m o y Fi pasa r 5 e le j E te So E eae M m PA Match symbols matched 60 urenatehed 131 Match symbols matched 39 uromatched 2 sampled vl x1 Raw 2 D Image e sample ya ul Raw 2 D0 Image dio Fig 5 8 Matched spots are marked with letters unmatched spots are circled If multiple unmatched spots appear in certain areas of the gels review these areas using the Match gt Show Offsets F7 command and add more landmarks to facilitate auto matching 5 3 a Editing MatchSets The commands for editing a MatchSet are located on the Match gt Edit Matches submenu or the Match gt MatchSet tool The Edit MatchSet dialog is similar to the Create MatchSet dialog See 5 1 Creating a MatchSet The exception is that there is no Close original image windows after creation option 5 12 Matchsets i Create MatchSet on disk a a x Mame New Match Auto Description Comments MatchSet level Member sharing f Level 1 members are gels f Do not make copies refer to the originals Higher level members are MatchSets Copy the onginals into this matchset
214. d the spot centers will appear marked with crosshairs Review the results of spot detection by studying the crosshairs on the image The number of spots detected is displayed in the Spot Count field Li Spot Detection Parameter Wizard a t igy espai maw E bl Bie lo le Image samples v1 Filtered Parameter Set Matts par Load Save Detecting and Editing Spots Basic Advanced Step 1 Give Manual Guidance al Click on a faint spot a Click on a small spot if different from the faint spot LA Box the largest spot cluster Help Step 2 Test Settings Sensitivity 0 67 e Size scale a e 2 Find Spot Centers Min soak fass 4 y Spot Count 334 _ Gaussian model during test takes longer Help Optional Controls Streaks Background Smoothing Speckles x Apply Sypro Ruby speckle filter Help Sensitivity 50 00 e Find Speckles Speckle count f 066 Show Speckles a Si Fig 4 4 Spot crosshairs marked in the Spot Detection Wizard If all the spots you want to identify have been detected you can save this parameter set If significant spots have been missed you can change the faint small and large spot selections in Step 1 or change the Sensitivity Min Peak and Size Scale directly Note Adjustments to these settings will automatically redetect spots Sensitivity If significant faint spots in the scan have been missed you can use the Faint Spot t
215. de assumes you have a working knowledge of your computer operating system and its conventions including how to use a mouse and standard menus and commands and how to open save and close files For help with any of these techniques see the documentation that came with your computer This guide uses certain text conventions to describe specific commands and functions File gt Open Choosing the Open command under the File menu Dragging Positioning the cursor on an object and holding down the left mouse button while you move the mouse CTRL S Holding down the Control key while typing the letter s Right click Clicking the right mouse button Left click Clicking the left mouse button Double click Clicking the left mouse button twice Some of the illustrations of menus and dialog boxes found in this manual are taken from the Windows version of the software and some are taken from the Macintosh version Both versions of a menu or dialog box will be shown only when there is a significant difference between the two PDQuest User Guide 2 Overview of Raw 2 D Gel Electrophoresis Raw 2 D electrophoresis is a method for separating proteins and nucleic acids in a sample into a two dimensional pattern of spots in a gel It combines the techniques of isoelectric focusing IEF with SDS polyacrylamide gel electrophoresis SDS PAGE Since these two separation methods rely on independent properties of proteins chemical and physical
216. dix K VersaDoc 1 Select the application 2 Position and focus the object to be imaged 3 Set the exposure time 4 Acquire the image K 2 Step Select Application In Step 1 you select the appropriate filters and other imaging parameters for the type of gel blot plate or other object that you are imaging Each set of parameters you select is called an application The VersaDoc supports multichannel sequential imaging This allows you to automatically image the same object using up to four different applications e g to detect different types of stains on the same gel First you select a channel then you select the application under that channel K 2 a Selecting a Channel Note Illumination Flat Fielding will be disabled if you use multiple channels The four channels are accessed using the tabs under Step 1 Channel 1 is always enabled that is the VersaDoc will always capture an image using the application settings selected under Channel first To enable any of the remaining channels click on a channel tab then select the Enable Channel checkbox and select the application for that channel as described below Enabled channels have a green check mark on their tabs Note If you use the Optimize Exposure feature only the application under the selected channel tab will be imaged other channels even if enabled will be ignored You do not need to enable Channels 2 4 in sequence For example you can set
217. dows or shift click the Macintosh Images in a scanset or a MatchSet will be loaded into the same window Click the Parent Folder button to go up one level in the directory tree Click the Desktop icon to open the desktop folder In the Look In field click the default name PDQuest User Guide or down arrow to the right of the field to display a hierarchical tree structure locating the default drive folder and file Note The drive buttons only include mapped drives If you need to access an unmapped network drive you can type the drive name directly into the Folder field using UNC notation e g remote server name shared directory name file name Or you can create a shortcut to the drive on your desktop To go directly to the folder where your images are stored click the Sample Images Folder To go to the folder containing your MatchSets click Sample MatchSet Folder Specify the locations of these folders in Path Preferences under Edit gt Preferences An image created in the Windows version of PDQuest can be opened in the Macintosh version and visa versa However you must add the appropriate extension to your Macintosh files to open them in Windows e g gsc for Raw 2 D scans gim for Filtered images csc for calstrip scans etc PDQuest comes with a selection of sample images In Windows these files may be found in the Discovery Series Sample Images 2D directory On the Macintosh they are stored in the Sample Image
218. e Analysis Set Manager Spot Review Image Stack EE Scatter Plot Ei al Annotate Bal Browse annotations eal More Annok Tools i I Mrpl Standards Spot Mumbers Plot SSP Grid Ctrl F9 HalfStock Master Display SSPs lt F9 Display All SSPs 1307 1311309 27217230A 82 gt a Transfer 55Ps 2304 Pan 730 Assign DSM Grid ere o OS Plot DSM Grid a 2203 1 1 a1 221 pose 221 1 Plot OSM Grid Counts Display DSNs a pla yore 422 2204 5942 Display All Dos 1220 2206 2208 Transfer DSMs 221 SSF mmber 201 22u3 Halfsi 140 Master E mu Fig 6 24 Spot number submenu With the MatchSet open and active select Plot SSP Grid CNTRL F9 from the submenu to display the SSP grid on the Master Numbered vertical and horizontal grid lines will appear on the Master The grid is helpful for locating spots by SSP number However it is an approximation and should only be used as a guide If enough spots are added to a single grid block to use up the 99 available numbers numbers will be assigned from other blocks Display SSPs displays SSP numbers next to every spot in the active window You may need to zoom in on the image to be able to read them 6 33 PDQuest User Guide Display All SSPs displays SSP numbers next to every spot in every open window Transferring SSP Numbers in a higher level MatchSet Transfer SSPs is used to copy the SSP numbers from one higher level MatchSet member to the ot
219. e In this mode an acquisition window will open and the controls will appear active but PDQuest User Guide instead of capturing real images the window will create dummy images of manufactured data You do not need to be connected to an imaging device to open a simulated acquisition window This is useful for demonstration purposes or practice scans To enter simulation mode hold down the CTRL key and select the name of the device from the File menu The title of the acquisition window will indicate that it is simulated Note There is no simulated calibration for the GS 700 and GS 710 Imaging Densitometers E 1 GS 710 Acquisition Window To acquire images using the GS 710 go to the File menu and select GS 710 The acquisition window for the densitometer will open displaying a control panel and a scanning window Appendix E GS 710 Densitometer GS 710 Transmitted Intensity Fig E 2 GS 710 acquisition window The scanning window is marked by grid lines that divide the area into square centimeters These are numbered 1 29 top to bottom and 1 21 left to right if the light source is reflective and 1 23 top to bottom and 1 20 left to right if the light source is transmissive see below To hide the gridlines click on the Hide Grid checkbox under Options Below the main scanning window is the calibration strip window Every time the densitometer calibrates an image of the calibration strip will ap
220. e You can also trim unneeded parts of an image to reduce its memory size See section 3 13 Cropping Images Note This command only works on Raw 2 D scans This function is comparable to scanning at a lower resolution in that you are increasing the size of the pixels in the image thereby reducing the total number of pixels and thus memory size Note Reducing the file size of an image will result in some loss of resolution In most cases this will not affect quantitation In general as long as the pixel size remains less than 10 percent of the size of the objects in your image changing the pixel size will not affect quantitation Select Image gt Reduce File Size The dialog box shows you the size of the pixels in the image Pixel Size X by Y microns the number of pixels in the image Pixel Count X by Y pixels and the Memory Size of the image As you increase the size of the pixels the pixel count will decrease as will the memory size You can increase the pixel size in either dimension see the following figure for an example You cannot reduce the pixel size you can only increase it You can reduce the pixel count 2 18 General Operation Before TORNIEN x Confirm reduce resolution Ready to reduce sampleb v1 1 Raw 2 0 Image to 300 49 189 39 micron resolution The image size Will be 304 766 pels Reduce Copy and Reduce Cancel Pixel size increase confirmation Reduce File Si
221. e X ray film Blue film Linear Coomassie Blue Silver stain Gel Copper stain Zinc stain Stain all Positive dark bands light background Photograph Negative light bands dark background Coomassie stain Colloidal gold stain Amido black stain Blot AP Substrate BCIP NBT HRP Substrate 4CN HRP Substrate DAB Fig F 4 Applications available in the GS 800 acquisition window PDQuest User Guide Choosing Your Own Settings If you know the filter and light source settings you want or want to experiment with different settings you can choose them yourself Filter Red x Green jBlue hite Light Reflective e Transmissive Fig F 5 GS 800 Custom Application controls Next to Filter click on either the Red Green Blue or White checkbox or a combination of two of the first three Red Green Green Blue Red Blue Filter Color Wavelength Application Examples 595 750 nm Coomassie G 250 BCIP NBT Fast Green FCF Methylene Blue 520 570 nm Coomassie R 250 Basic Fuchsin 400 530 nm Crocein Scarlet 400 750 nm Silver Stains Copper Stains Film Fig F 6 Examples of filter colors and applications Next to Light select the appropriate light source e Select Reflective for scanning opaque mediums such as dried gels on filter paper arrays or photographs The scanning dimensions in this mode are 30 cm x 40 cm e Select Transmissive for scanning transparent mediums such as films gels
222. e UV on fluorescent gels UV light is only available for the Fluorescent spot cutters Step Image Acquisition Acquire Image Light on Gel Light off PDF UY on Fluor Tinmelzecs Fig 9 10 Light settings If UV light is selected specify an exposure time in seconds in the field Also make sure that the UV lamp switch on the back of the cutter box is on Note For Sypro Ruby stains an exposure time of 1 30 seconds is recommended fin Acquire image When you have selected a light option click Acquire Image 9 24 Integrated Excision Tool Note For UV exposures there will be a 15 second delay while the lamps heat up before the exposure begins The camera will take a single image of the gel or membrane on the platform then a background image to adjust for image background When the exposure is complete the image appears in the window y To adjust the brightness and contrast of the image open the Transform window by clicking on the button to the right of the Image window You can also use the Transform button on the main toolbar Step 2 Positioning the Data Area Box You will be prompted to position the green data area box that surrounds the camera cutter image a PDQuest xj Step 2 Position the green data area box 1 lf incorrect orientation flip and or rotate 2 Drag and drop the data area box in the camera image right hand pane o that t encloses exactly the
223. e aa 2203 13 peptides 13 88 coverage 116 35kDa ci protname 2204 score 20556 8 BETA GALACTOSIDASE EC 3 2 1 23 LACTASE m 2205 ESCHERICHIA C 13 peptides 13 88 coverage 116 35kDa 5 4lpH 2206 Add Edit Delete Std dci 20990 3 2301 CVUS9933 NID gZ190711 Cloning vector pBlI C L Show description 13 peptides 13 79 coverage 117 03kDa 5 66pH 2302 score 20417 1 Image Options 2303 CvVU46439 NID gl245106 Cloning vector placz 13 peptides 13 47 coverage 120 0lkDa 5 58pH x Highlight target spot 2304 score 19952 2 Mark all annotated spots Format 2401 x Mark spots in category Format Sot SSP Name C Show spot annotations Format x Auto select same entry gt Select spots in image J Apply ent Close E Help Fig 10 1 Example of the Hit Table annotation category and entries In addition the protein name is included in the standard protname category which will be created if it doesnt already exist so you can sort the spots by protein name in the Select Spot list Note Each category is specific for the mass spec instrument used For example if you import information from both MALDI and MS MS you would create both a Hit Table MALDI category and a Hit Table MS MS category 10 3 Mass Spec Score Overlay If you have imported mass spec data for spots using Import MassLynx Results you can display overlays indicating the confidence le
224. e an sbk extension Macintosh backup files will have the word backup after the file name Note To open a Windows backup file you must first change the file extension To locate the file after changing the extension select All files in the Files of type drop down list of the Open dialog Highlight Saturated Pixels When this box is checked any saturated pixels in the image will appear highlighted in red in the scan window and in the pop up image window To view hide saturated pixels in the pop up image window use the Image gt Transform command Hide Grid To hide the gridlines in the scanning area window click on the Hide Grid checkbox D 15 PDQuest User Guide D 16 Appendix E GS 710 Imaging Densitometer Fig E 1 GS 710 Imaging Densitometer Before you can begin scanning images with the GS 710 Imaging Densitometer your instrument must be properly installed and connected with the host computer See the hardware manual for installation startup and operating instructions PC Only A Note About SCSI Cards The GS 710 is connected to your computer by a Small Computer System Interface SCSI cable To use the GS 710 you must have a SCSI card installed in your PC If you have a PC with a Windows 98 or Windows ME operating system you may also need to load the SCSI and WinASPI drivers that came with the card Simulation Mode Any of the imaging device acquisition windows can be opened in simulation mod
225. e f stop on the camera to the lowest setting 1 e the maximum aperture This reduces the depth of field allowing you to more accurately focus the camera Then after focusing increase the f stop to the K 7 PDQuest User Guide desired setting See Table 1 Recommended Exposure Times and Lenses on page 9 After you have positioned your sample click on the Focus button and look at the image in the acquisition window while adjusting the focus on the camera lens While focusing the camera will limit its focus to a small portion of the sample this will not affect any zoom lens adjustments you may have made See the VersaDoc User Manual for details on focusing When you click on Focus the light inside the camera box automatically switches on To turn the light off while positioning deselect the Light On checkbox When you are finished focusing click on the Stop button K 4 Step Ill Set Exposure Time The exposure time is the period of time an image is integrated on the CCD The effect 1s analogous to exposing photographic film to light Setting an Exposure Time Different applications have different optimal exposure times If you are imaging using multiple channels you can select a different exposure time for each channel First select the channel using the tabs then select the appropriate exposure time for that channel See Table 1 Recommended Exposure Times and Lenses on page 9 You can enter an expos
226. e fields are used to specify the size of your imaging area in centimeters which in turns determines the size of the pixels in your image 1 e resolution When you adjust one imaging area dimension the other dimension will change to maintain the aspect ratio of the camera lens Note Your imaging area settings must be correct if you want to do 1 1 printing These are also important if you are comparing the size of objects e g using the Volume Tools between images Auto Exposure Threshold When you click on Auto Expose the exposure time is determined by the percentage of saturated pixels you want in your image This field allows you to specify that percentage Typically you will want less than percent of the pixels in your image saturated Consequently the default value for this field is 0 15 percent Reminder When this checkbox is selected the software will warn you to turn off your transilluminator light when you exit the Gel Doc acquisition window or when your system is idle for more than 5 minutes Video Printing Footer Information The checkboxes in this group allow you to specify the information that will appear at the bottom of your video printer printouts Save Options To automatically create a backup copy of any scan you create select the Make Backup Copy checkbox A 13 PDQuest User Guide Note To open a Windows backup file you must first change the file extension To locate the file after changin
227. e maximum and minimum pixel values within the filtering window e Power Mean This filter is useful for suppressing salt and Gaussian noise within an image it worsens the effect of pepper noise It replaces the value of the pixel being processed with the power mean of the pixel values within the filtering window e ContraMean This filter is useful for suppressing pepper and Gaussian noise within an image it worsens the effect of salt It replaces the value of the pixel being processed with the contra harmonic mean of the pixel values within the filtering window e Adaptive This filter is useful for suppressing Gaussian noise and salt and or pepper within an image If your image contains a mix of salt and pepper select this filter To begin filtering select a filter type from the pull down list A pop up box will ask you to select a filter size hi PO QUEST A Select filter size Select a size that is larger than the average note feature as PRP 9x9 Cancel Fig 3 23 Selecting a filter size The available filter dimensions range from 3 x 3 pixels to 9 x 9 pixels See the previous section for guidance on selecting a size Because filtering is an irreversible process a pop up box will give you the option of filtering the original image creating a copy of the image to filter or cancelling out of this operation 3 37 PDQuest User Guide If you choose to copy and filter you will be asked to enter a n
228. e must use one of these two delimiters in order to be imported Relate Column to Category This field shows a list of columns in your text file Whatever is in the first line of your file appears in parentheses next to the column number To relate a column to a category highlight the column in the Relate column to category field and click the down arrow to select a category To add a new category click the Add button See spot Annotation Category Editor for more information Any column that does not have a category relationship will not be imported If the Show description box is checked the category description appears beside the category name Uncheck this box if you do not need to see the category description Save Settings The save settings button allows you to save the import settings of the Import annotations dialog The save settings option saves First line header and Confirm overwrite selections the delimiter type and the column to category relationship for each column Saving the import annotation settings is useful tool especially if you plan on adding many annotations from different files The Save Settings option saves your settings in a simple text file Load Settings To load a saved settings file click the Load Settings button and locate the settings file you wish to load Click OK 7 30 Analysis Sets and Annotations Click Import when you are ready to import your annotations A progress box appears indicating the nu
229. e program The software will open and the Software License screen will be displayed Note If you attempt to start the downloaded program and receive an Unable to obtain authorization message you will need a Hardware Security Key to run the program Contact Bio Rad to obtain a key Software License Welcome to POG uest 30 day Free Trial Free Trial Allows you to fully use the package to create and analyze the images of pour choice You will need BEE to fill out the Registration Form and submit it to Bio Rad Enter Password Basic mode Allows you to acquire new images open existing images Registration Forn perform basic editing functions print export to TIFF and Save Click Help for further information Contact Exit POG uest Your local Bio Rad office for information on purchasing a full user license or visit us on line at www bio rad com Help Fig 1 12 Free Trial screen In the Software License screen click on the Free Trial button This will open the Software License Registration Form Enter the required information you will not 1 16 Introduction have a purchase order number or software serial number and can leave these fields blank and click on Submit Via Internet A free trial password will be automatically downloaded to your computer This password will allow you to use the software for 30 days If you decide to purchase the software during that period contact Bio Rad to
230. e root name only with no exposure number H 6 c Save Auto Save After Scan To automatically save any image you create click on the Auto Save After Scan checkbox Note In PDQuest this option is preselected and cannot be turned off All images must be automatically saved when acquired With this checkbox selected when you click on Acquire or Live Acquire a Save As dialog box will open asking you to specify a file name and location for the image you are about to create The scan will begin when you click on the Save button Note that in Live Acquire mode you can save your intermediate exposures by selecting Auto Save After Scan and then Save All Intermediate Images Make Backup Copy You can automatically create a backup copy of any scan you create To do so first select Auto Save After Scan see above then select the Make Backup Copy checkbox With this checkbox selected when you save a scan a backup copy will be placed in the same directory as the scanned image Windows backup files will have an sbk extension Macintosh backup files will have the word backup after the file name Note To open a Windows backup file you must first change the file extension To locate the file after changing the extension select All files in the Files of type drop down list of the Open dialog H 6 d Imaging Area Size The imaging area is the area of the sample that is captured by the camera and displayed in the scan window To
231. e selected in the ASM become inactive To view these sets in the Analysis set Template check the box labeled Display Selected sets 7 1 e Statistical Analysis Sets Statistical analysis sets are composed of spots from either replicate groups or classes See Section 6 6 Sample Database for information on classes found to be significant according to the statistical test that you specify To create a statistical analysis set select Statistic from the Select Set Type dialog 7 12 Analysis Sets and Annotations Ll Analysis Set Template Matchset HalfStock 3 E nA x Description x Mark spots x Repl Gels E Stock _ Display selected sets Compare f Classes E Han B Mark spots on e Master gel Method Student s t test _ Log transform Mann Whitney Signed Rank Test T able Show g Wwilceoson Paired S ample Test Table l 2 Window labels Significance Level in 90 95 98 99 55P number Operand gels All gels Format Spot court 745 af Save X Cancel 2 Help Fig 7 5 Statistical Analysis Type Statistical analysis sets only work with replicate groups or classes because the tests require multiple gels Enter a name for the analysis set at the top of the dialog box Note If you have Normalization enabled normalization parameters will be used for spot quantities See Section 6 7 Normalization for further information Select whether you want
232. e selected spot to all the gels Clicking Auto Add will add the spot marker to the gels where it does not appear A red circle indicates where the spot marker is added Auto Remove removes the selected spot from the gels Auto Cancel Cancels the spot in the group This does not remove the spot from the list but it does remove it from your analysis Manual The Spot edit actions allow you to manually add or remove cancel or restore combine or un combine spots on the images in the replicate group Click update under the Consensus Summary table for the changes to take effect When you have finished making your changes to the consensus groups click Update under the Consensus Summary table Analysis Tools 6 5 c Global Actions Global actions are much the same as the adding and removing of individual spots but are performed on all the spots in the cell Auto Add All allows you to automatically add all the spots of the selected cell to all the gels For instance a replicate group of three gels may contain 15 spots located in two of the gels Clicking Auto Add All will add the spots to the gels where they do not appear Auto Cancel All Cancels all the spots in the cell This does not remove the spot from the list but it does remove it from your analysis Auto Remove All removes the selected spots from the gels Since the global actions affects multiple spots on multiple gels PDQuest warns you before proceeding with t
233. e the cutter is calibrated you do not need to repeat the procedure during normal day to day use You must recalibrate if e The instrument is moved e You install or remove the lens filter after calibration 9 2 c Cutting Spots After you have focused the camera and calibrated the spot cutter you are ready to begin cutting spots The control panel is designed to guide you through the spot cutting procedure 9 17 PDQuest User Guide i Integrated Excision Tool lps ed Cut list Analysis name Dermo Gual 5d Gel spots to cut 10 Analysis set spot count 12 Total cuts incl multi 10 Cut MTP SSP Gel Quantity Run Cuts Cut date Status LJ 1 401 4202 Derma s0deqnee 3204 5 05 Dec 2002 Completed 2 L 1 402 1303 co HE St 3509 5 05 Dec 2002 Completed x 1 403 8201 Demo 5Odegree2 2567 1 x 1 404 5401 Demo 30degreez 4155 7 x 1 405 3701 Demo 5Odegree2 6490 9 x 1 406 0602 Demo 3Odegree2 15572 x 1 407 0703 Demo 5Odegrees 2065 6 x 1 408 4401 Demo 5Odegrees 2491 9 x 1 409 aro4 Demo 5Odegrees 2560 3 x 1 410 7303 Demo 5Odegrees 2649 3 Status Wash options Station X Station 2 x Station 3 X Station 4 Member image 1 1 1 1 1 1 Cutter image 1 1 1 1 Mise Options x Multiple cuts spot Cutter options X Spin when cutag L Cut longer 2 Spin when ejecting X Double eject _ Save validation image Setup HAUS Ae
234. e the mouse button the border changes to a dashed blue line indicating a selected area To reposition the scanning box you have selected position your cursor inside the box and drag The entire box will move To resize the box position your cursor on a box side and drag The side you have selected will move To redo the box entirely position your cursor outside the box and drag The old box will disappear and a new box will be created Appendix I Personal FX You can also select the scanning area by entering coordinates in the appropriate fields Top Bottom Left Right After you enter a coordinate the position of the scanning area box will change accordingly When selecting be sure to include the entire area of interest and be generous with borders You can always crop the image later 1 3 Step Il Select Resolution The Personal FX acquisition window allows you to scan at 50 100 200 or 800 micrometers These resolutions are listed as option buttons in the control panel Step Il Select Resolution 50 micrometer f 100 micrometer gt 200 micrometer gt 500 micrometer Image file size 6 09 Mb Fig l 4 Resolution option buttons The resolution you select should be based on the size of the objects e g bands spots you are interested in For example e 50 micrometer resolution should be reserved for images requiring the highest level of detail e g high density in situ samples 1 536 well micro
235. e to a better understanding of cell growth and regulation Xi PDQuest User Guide Sample Experiments Si Valuable information can be learned by exposing cells to a set of specific experimental conditions and subsequently examining their biological response A preliminary in vitro experiment that is useful when beginning Raw 2 D gel work is to radiolabel a cell s proteins to steady state with 35S methionine Running gels of these proteins will help to determine the commonly expressed proteins in the cell under normal conditions Subsequently other amino acids such as 3H leucine 3H proline 3H lysine etc can be used to radiolabel the proteins to steady state This will label any proteins that do not contain methionine and were therefore not detected in the first experiment and will provide preliminary information on the amino acid composition Other experiments complementary to Raw 2 D gels include cell fractionation procedures and post translational modifications phosphorylation methylation etc Data from these experiments can be added to a database accumulating information about these proteins Bio Rad Listens The staff at Bio Rad are receptive to your suggestions Many of the new features and enhancements in this version of PDQuest are a direct result of conversations with our customers Please let us know what you would like to see in the next version of PDQuest by faxing calling or e mailing our Technical Service
236. eckbox is selected the software will attempt to correct for minor systematic errors inherent in scintillation counting pipetting and exposing films The normalization process is based on the assumption that these minor errors affect all proteins equally It also assumes that few protein spots change within the experiment or that when changes do occur they do not alter the random distribution from one sample to the next When you select this checkbox a pop up box will display the calculated compensation value for each MatchSet member These values will also appear in the in the table at the bottom of the Normalize dialog 6 28 Analysis Tools 6 8 Mrpl Data In a MatchSet you can enter the molecular weight and isoelectric point values for your known protein spots With this data PDQuest can calculate MrpI values for all the spots in the MatchSet The functions needed to enter and display MrpI data are found in the Analyze gt MrpI Standards submenu and the MrpI Tools toolbar Analyze Identify Report Y Replicate Groups Group Consensus Describe Samples be Normalize Mr pl Tools E FirpI Lats Mrpl HrpI eee Analysis Set Manager Met D Ok E Spot Review Image Stack Scatter Plot Annotate Browse annotations More A4nnot Tools Mrpl Standards Enter Mrpl Data Mark Mrpl Spots Display Mrpl Data Plot Mrpl Grid Spot Numbers k Remove MrpI Data Transfer Mrpls Fig
237. ed the results of auto matching you are ready to identify the significant spots that could not be auto matched To highlight these spots in a member gel use the Mark Unmatched Spots command Shift F8 Match gt Edit Matches submenu in the gel s subwindow Unmatched spots are marked with red circles Use the Zoom Box tool to study each unmatched spot in detail in each gel If a spot was missed during spot detection you must add it to the Gaussian image then match If a spot was not included in the Master you must add it to the Master then match If an unmatched spot is not significant you can cancel it 9 30 Matchsets Otherwise if auto matching has missed a spot that should have been matched you can landmark it to improve the alignment and modeling of the Master or you can manually match it Note Manual matching will not change the modeling of the Master and therefore will not facilitate additional matching Select Manual Match from the Match toolbar or Match gt Edit Matches F submenu and click on the spot in the Master then click on the same spot in all the member images where that spot appears The spot will be marked with a purple box to indicate that it has been manually matched To unmatch a manual match select Manual Unmatch from the toolbar or Cl i submenu and click on the manually matched spot in a member image Unmatched Spots Choose Mark Unmatched Spots Shift F8 to circle spots you have been unable
238. elect Spots gt Show All Quality Note Spots of low quality may be more accurately quantitated using the spot boundary tools section 4 6 Spot Boundary Tools Spots created using the boundary tools are not assigned a quality value Low Quality Spots You can identify a set of spots that do not meet a threshold quality level You can then flag those spots as low quality and or redefine them using the spot boundary tools section 4 6 Spot Boundary Tools From the Spots gt Low Quality Spots submenu select Define Low Qual Set then click in the MatchSet or scanset Gaussian image In the pop up box enter a threshold quality value All spots below this threshold will be flagged as low quality When you click OK the low quality spots will appear highlighted in the image and the number of low quality spots will be displayed at the bottom of the image window To re highlight the low quality spots in the set select Spots gt Low Quality Spots gt Mark Low Qual Spots and click in the image 4 27 PDQuest User Guide To cancel all low quality spots in the set select Spots gt Low Quality Spots gt Cancel Low Qual Spots and click in the MatchSet or scanset Gaussian image To restore the cancelled low quality spots select Spots gt Low Quality Spots gt Restore Low Qual Spots and click in the MatchSet or scanset Gaussian image 4 12 Spot Parameters The Spot Parameters information box provides a variety of information about
239. elect a predefined colormap click the Load button From the list displayed click the set of colors you want to apply amp Color Palette i PDQUEST Colormap Hame coomass Select Color Preferencez Load Delete A Color Group Light Overlay Colors BEIGE COOTLASS Standard FALSE GRAY SILVER User defined sets of colors gt 3 gt serdefined Promace Standard Cancel Fig 3 11 Selecting a Colormap To create your own colormap adjust the colors within the color groups as described above and type in a new colormap name Click OK to apply your changes To remove a colormap click the Delete button Select the colormap to be deleted from the displayed list A pop up box will ask you to confirm the deletion If you want to return to the standard colormap for the selected group click the Reset button All colors will be returned to their default values 3 15 PDQuest User Guide 3 10 Multi Channel Viewer You can use the Multi Channel Viewer to distinguish different types and levels of fluorescence in a gel that has been imaged at different wavelengths The Multi Channel Viewer can be used to the merge the information from up to three different images of the same gel Note The viewer requires that the images being compared are exactly the size and in exactly the same position When capturing the images you should be careful not to move the gel between exposures If your im
240. elow the menu bar It includes buttons for the main file commands Open Save Print and essential viewing tools Zoom Box Grab and buttons to open the secondary toolbars See Fig 2 1 Main toolbar Window Help Print the selected window x Status Boxes id lt Reduce File Size Tool Help Graph Tools Fig 2 2 Tool Help and Status Box Tool Help If you hold the cursor over a toolbar icon the name of the command will pop up below the icon This utility is called Tool Help Tool Help appears on a time delay basis that can be specified in the Display Preferences dialog box under Edit gt Preferences You can also specify how long the Tool Help will remain displayed 2 2 General Operation 2 1 c Status Boxes There are two status boxes in PDQuest These appear to the right of the main toolbar The first box displays any function that is assigned to the mouse see section 2 8 Mouse assignable Tools If you select a command such as Zoom Box the name and icon of that command will appear in this status box and remain there until another mouse function is selected or the mouse is deassigned The second status box is designed to supplement Tool Help see above It provides additional information about the toolbar buttons If you hold your cursor over a button a short explanation about that command will be displayed in this second status box 2 1 d Secondary Toolbars PDQuest has secondary toolbars
241. els in the resulting image are larger in the horizontal dimension than in the vertical dimension This is useful for gels with bands where you are more interested in resolving in the y dimension to determine the size of bands and the spacing between them Specifying Your Own Resolution If you select Oversample under More Options see section D 7 for details you can specify your own resolution within the range of 43 169 microns micrometers With Oversample selected enter values directly in the fields next to X resolution and Y resolution in the main acquisition window D 8 Appendix D GS 700 Step lll Select Resolution resolution E D Microns Select Y resolution 50 of Microns Image file size 0 10 Mb Fig D 8 Entering a custom resolution with Oversample selected Image File Size Image File Size under Select Resolution shows the size of the scan file you are about to create If you do not have enough computer memory for the specified file size an error message will appear when you attempt to scan If this happens select a lower resolution or decrease the size of the area to be scanned Macintosh users can also increase the application memory partition See your Macintosh computer documentation for guidance D 5 Acquire the Image If you want to calibrate your scans read the following section on calibration before scanning To begin to scan click on the Acquire button The scanned image will be
242. ely will not change unless you have regular access to both types of spot cutters There are slight differences between the two types For instance because the ProteomWorks Plus spot cutter has a larger platform and cutting tip range it is not necessary to crop the left margin The Proteom Works Plus spot cutter also allows you to cut backed gels See Step 3 Set Cutter Options on page 8 16 Microtiter Plate Specify the microtiter plate size you are using by clicking on 96 wells or 384 wells Specify the order in which you want to the load the wells on the microplate by selecting Al A2 A3 or Al BI Cl This function is useful for matching a partial plate to the load order of a digest robot Well A1 position The fields and buttons in this section are used to calibrate the position of the microplate for proper loading of samples The number fields contain the X and Y coordinates in millimeters of well Al on a plate in the spot cutter plate holder Once you have set the position of this well the cutter robot will automatically calculate the positions of the remaining wells To set the well Al position position your cursor in the number in the X or Y coordinate field click to place the cursor in the number field and press Enter on your keyboard The tip will move to that position without attempting to lower into the well If the tip is not centered over the well Al on the microplate you will need to adjust the numbers in e
243. emplate grid pattern supplied wath this system The grid should be placed on top of the cutting mat which should be installed on the cutter first Make sure the grid is flat and aligned wath the stage edges nce you have placed the end on the cutting mat press Ready to proceed If you have performed this step before you may skip to Step 3 although this is not recommended Read Skip Back Cancel Help Fig 9 6 Spot cutter calibration step 2 The third dialog requires the calibration template grid pattern supplied with the PDQuest system Place the grid on top of the cutting mat which should have been previously installed on the cutter Make sure the grid is flat and aligned with the stage edges Click Ready to proceed You may also skip this step return to the previous dialog cancel the action or ask for help The fourth dialog prompts you to place the coomassie stained PVDF membrane on the mat Click Ready to proceed with cutting Back to return to the previous screen or Cancel to cancel the action At any point in the procedure you can move back to repeat a step 9 16 Integrated Excision Tool Note Calibration creates several parameter and image files in the Install prm directory in the Discovery Series directory on your hard drive These files should not be moved deleted or altered or calibration will be affected When calibration is complete you can proceed with normal use of the spot cutter Onc
244. en entered you will want to apply it to the 2 D scan and the calstrip scan To do so click on the Apply button If you want to apply different calibration data to your 2 D scan than to your calstrip scan if for example your 2 D scan and calstrip scans use different isotopes use the Apply Here button to apply the gel record to the currently displayed image only 2 D or calstrip scan Copying Gel Record Information If you have scanned several exposures of the same gel most of the calibration data will be the same for all of them except for the exposure start date and length Instead of entering all the information for each exposure you can enter it once and then copy it between exposures using the Copy button Clicking on Copy will open a list of available gel records Select the record that you would like to apply to the current image and click the mouse Click on the Cancel button to exit the operation L 1 c Sampling the Calibration Strio Segments The next step in calibrating your gels involves sampling the densities in the segments of the calstrip to establish a correlation between the sampled intensities recorded by the scanner and the counts entered into the Calstrip Record If you have not already done so display the calstrips If you have gels with multiple exposures use the Configure Calstrip Windows function to automatically divide the screen into enough windows to display all the calstrips for a given gel Flip Ca
245. en this box is checked any saturated pixels in the image will appear highlighted in red in the scan window and in the pop up image window To view hide saturated pixels in the pop up image window use the Image gt Transform command Hide Grid To hide the gridlines in the scanning area window click on the Hide Grid checkbox PDQuest User Guide Warn on Saturated Pixels The Warn on Saturated Pixels option is checked by default Uncheck this box to disable this warning Appendix J Molecular Imager FX Family FX Pro FX Pro Plus and Molecular FX Fig J 1 Molecular Imager FX Before you can begin acquiring images using any of the Molecular Imager FX family of products the particular instrument must be properly installed and connected with the host computer See the corresponding FX hardware manual for installation startup and operating instructions J 1 PDQuest User Guide Note The FX should be turned on and the initialization sequence completed before the host computer is turned on except in the case of certain Power Macintosh configurations See the hardware manual for more details PC Only A Note About SCSI Cards The FX is connected to your computer by a Small Computer System Interface SCSI cable To use the FX you must have a SCSI card installed in your PC If you have a PC with a Windows 98 or Windows ME operating system you may also need to load the SCSI and WinASPI drivers that came with the card Simu
246. enter a new name for the group in the pop up box 6 10 Analysis Tools Quantitating a Replicate Group After you have created a replicate group you can calculate the average spot quantities in the members of the group by selecting Spots gt Show Quantity F6 Place your cursor in each image for which you want to see spot quantity Quantity will not display for the Master image Spot quantity displays as blue numbers with a text box stating whether the quantity is normalized 1 Matchset MSO0002 2001 10 04 561 6 aT 65466 ee 113821 als 7 RAAS Ss Prize oer a Zio OZE 50349 Sigzearest tL owt TES pe 97217 amar 82674 16 1 454564 1357918205880 4 97315 Un norm spot quan 221605 sample4 vl xl Raw 2 D Image a se 4 7148742523 B Sen 71103640 a E It io 20949 ne AMRASN a ae A 1182563 3655 Hy Mat mT E s te 83293 Zn E ge a ae otoce xs aegi0re i oe 6 Ls le 185626 8 AS er 39 As 66672 3 3 tens q _ Boruu ES 432 mu ut 1 foveuu 21 tI tee 3927 70404 9 230748 DIOIL ice leis ra a alo a 31707 32475272 7 age oF LOA TS wy fag 102198 Peres Un norm spot quan sample5 vl xl Ra 9546 353038 179 250784 e 178535 21 E IBARRA pees e547 232924 foiz e ae O7 172555 Binal 2312 RA F g EON 2 72 TS ae 74620133302 Meadas 43218 116 si 13694376 e 487331 q 4 223573 Un norm spot quan 3981 65 y samp
247. eplicate Group Spots Matched Match Rate 1 Match Rate 2 Corr Coeff Detect paramet gg Fstock1 v1 Stack 1285 1283 100 1 000 stack stock2 v1 Stack 1273 903 70 70 0 939 Ma stock3 v1 Stack 1309 942 71 73 0 929 Mia hall w1 Hal 923 712 77 55 0 895 stock test half v1 Half 236 G71 0s 52 0 680 Al E Master is marked with Correlation coefficient based on stack y Replicate group information Replicate Group Member Matched to all Mean CY 3 B22 stock 22 70 MatchSet information Spots matched to even member 520 Overall mean coefficient of al 20 12 variation of all replicate groups Half 3 562 17 45 wf Close E Normalize Rematch z Scatter Plot El Hep Fig 5 22 Matching Summary dialog Member information lists each gel in the MatchSet and information regarding each gel including total number of spots in the gel and the number of gels matched to the master The Match rate 1 column lists the percentage of matched spots relative to the total number of spots on the gel The Match rate 2 column lists the percentage of matched spots on the gel relative to the total number of spots on the master To change the referrence gel for the correlation coefficient double click the gel name in the list To view the log file for a gel click the log name in the gel table MatchSet Information MatchSet information contains information regarding the total number of spots matched to
248. er to view your data if it does not occupy the full intensity range of the image Log The Log checkbox changes the way the data is displayed in the histogram so you can better discern subtle changes in signal intensity Image Max Min Image Max and Min display the range of signal intensity in the image The image units are determined by the type of scanner used to create the image In the case of densitometers you can select Calibrated Quantity to display your image units in O D s 3 25 PDQuest User Guide Invert Display The Invert Display checkbox flips light bands on a dark background to dark bands on a light background and visa versa Once again the actual data will not change only the image Highlight Saturated Pixels When the Highlight Saturated Pixels checkbox is selected areas of the image with saturated signal intensity are highlighted in red This setting does not apply to Gaussian images 3 13 Cropping Images When matching spots between the gels in a MatchSet the MatchSet images should be all the same size and shape If the original scans of these gels are different sizes you will need to crop them before creating the MatchSet Note You can only crop Raw 2 D scans not Filtered or Gaussian images The crop tools can also be used to reduce the file size of images To reduce pa the file size of images by lowering the resolution see Reduce File Size Image Info Ctrl I Transform CErl B Image Tools
249. er of exposures Optimize Exposure xX Settings Total Exposure Time sec 10 0 Starting Exposure Time sec ji a Number of Exposures E _ Save Images Cancel OF Fig C 8 Live Acquire settings Note You should specify no more than 10 exposures in the Live Acquire Settings dialog to avoid excessive build up of image background in later exposures The C 8 Appendix C ChemiDoc XRS fewer the exposures the less background will be added to the image See the Release Notes for additional instructions on reducing background in images captured using Live Acquire Select the Save Images checkbox if you want to automatically save each exposure as it is taken Click on OK in the settings dialog to begin taking exposures If you selected Save Images a Save dialog box will open in which you can specify the base file name and location of the exposure files When you click on Save the exposures will be taken The specified number of exposures will be taken at equal intervals between the starting exposure time and total exposure time When each exposure is complete an image window containing that exposure will open in front of the ChemiDoc XRS window Note that the first exposure will have the base file name the default base file name is the computer user name and a time stamp Each subsequent exposure will have a version number v2 v3 v4 etc appended to the base file name The highest version number
250. erage intensity will be displayed in a pop up box 3 11 PDQuest User Guide Plot Density Distribution Plot Density Distribution displays a histogram of the signal intensity distribution for the entire image Select the command from the menu or toolbar and click on the image lta y Plot Cross section To display a cross sectional intensity trace centered on a point on the fe image select Plot Cross section from the submenu or toolbar or use the Ctrl T key command and click or drag on the image The trace will be displayed and will be continuously updated as you move your cursor over the image The intensity n ODs or counts at the cursor point is listed as is the maximum intensity along the lines of the cross section Note The sampling width for the density traces is one image pixel Density Toole Position m A ag E m iS mm k Quantity 0 32 0D Max Quant 0 49 OD Click button then click or drag on the image Fig 3 7 Plot Cross section tool 3 12 Viewing and Editing Images Plot Vertical Trace the point where you click your mouse Select the command from the menu Plot Vertical Trace plots an intensity trace down a vertical line centered on Ze or toolbar and click or drag on the image Note The sampling width for the density traces is one pixel 3 9 Colors Edit gt Colors opens a dialog box in which you can adjust the colors of the image windows buttons etc ad Color Palette E
251. es This opens the Pair Samples Dialog 6 21 PDQuest User Guide hi Pair Samples Pair Selections Unpair Selections Import uf Done H Help Fig 6 18 Creating Sample Pairs To create a sample pair highlight a sample inn the left column and a sample in the right and then click Pair Selections The Right sample turns red to indicate it is paired with the selected sample in the left column To un pair a selection highlight the sample pair you wish to un pair and click un pair selection If the sample you select in the left column is already paired to a sample pairing it with another sample will automatically un pair the selected sample with original sample You can also import sample pairs by clicking Import This opens the Select file to Import dialog Locate the file you wish to import and click Open The format of your file must be sample1 sample2 sample3 sample4 Each row in the text file represents a pair separated by a comma Upon importing the sample pairs PDQuest attempts to locate samples in your list which match the pair in the file If a match for a pair cannot be found it is simply not imported Only those pairs containing samples in your database are imported 6 22 Analysis Tools Note Importing sample pairs will overwrite any pair selections you have already created containing samples that match imported pairs 6 Normalization When comparing gels in a MatchSet there is often some variat
252. es in this group allow you to specify the information that will appear at the bottom of your video printer printouts B 15 PDQuest User Guide Save Options To automatically create a backup copy of any scan you create select the Make Backup Copy checkbox With this checkbox selected when you save a scan a backup copy will be placed in the same directory as the scanned image Windows backup files will have an sbk extension Macintosh backup files will have the word backup after the file name Note To open a Windows backup file you must first change the file extension To locate the file after changing the extension select All files in the Files of type drop down list of the Open dialog simple Acquisition Mode The Simple Acquisition mode option allows you to reconfigure the ChemiDoc acquisition window to simple mode In simple mode the acquisition window contains the same steps as the advanced acquisition window with the exception of Step IV Optimize display and Step V Analysis Manual exposure is also disabled in simple mode B 16 Appendix B ChemiDoc amp Chemi Doc Fig B 11 Simple Acquisition Window To change the acquisition window to Simple Acquisition Mode check the box marked Simple Acquisition Mode The change will take effect the next time you open the acquistion window PDQuest User Guide B 18 Appendix C ChemiDoc XRS Fig C 1 ChemiDoc XRS Before you can begin acquiring ima
253. es the total peak value for a saturated spot Note These options are only available for groups and classes Choose whether to compare gels replicate gels or classes See Section 6 6 Sample Database for information on classes Select the items you want to compare If you click A the pop up box displays a list available for analysis Select what you want to compare from the list and their names display in the A or B field Now select whether you want to highlight spots that were on in A off in B On in A or on in B off in A On in B Finally you need to tell PDQuest what turned on or turned off means How many times greater than the minimum detectable spot should the quantitation be for a spot to be included in this analysis set Should it be ten fold greater than the minimum If so enter 10 00 next to the Fold over minimum sensitivity prompt Click on the Save button at the bottom of the dialog box to add the analysis set to the ASM Be sure to enter a name for the set If you forget a pop up box will remind you to do SO As with all other types of analysis sets when the Analysis Set Template is open you can view the results in the MatchSet immediately To the right of the dialog you can specify the spot marker on which gel s to show the spots and what information to show on the gel s When you open the Analysis set Template whatever analysis sets you have selected in the ASM become inactive To view these se
254. eselect these options to turn off one or all of these functions To close the Spot Review tool window click Done 6 4 Analysis Tools 6 2 Image Stack Tool The Image Stack Tool allows you to scroll through the images in a scanset or MatchSet layered on top of one another Using this tool you can easily compare spots that appear disappear or change size in gels run under different experimental conditions You can flag these spots and save the flagset using the new Save Flag Spot Set command Note Your images should be close to the same size with spots in the same relative positions to use this tool With all your MatchSet or scanset images open select Image Stack Tool from the Analyze menu to open the stack tool i Image Stack Tool Matchset ahmed Use ctrl click to flag spots of interest shift ctrl click to un flag Select gels to display i seen cross means matched to master ee ared cross means must be added to master ahned Master ma i T sample3 vl Filtered ak 4 gt gt sanple3 vl Gaussian gt b sample3 vl xl Raw 2 D Image g P d gt vl Gaussian i gt e j A 4 z5 sample4 vl xl Raw 2 D Image 2 1O E y de if ns A a a e ALL Y fen 45 el j Ei e a 1 e al d PE gt ddi back a as Se g ll aybac X Show crosshairs Step KA LjAuto slow pee pene fast Image
255. et type dialog This opens the Arbitrary Analysis Set template i Analysis Set Template Matchset HalfStock ae xj Hame JArbit Description 5 Mark spots x Operand Member _ Display selected sets Mark spots on Master gel Method f No spots ae Add spots to set All spots in member Se Remove spots from set Saturated spots f Operand gels All gels Show Window labels _ 55P number Import list of SSP from text file Format Spot count o yf Save X Cancel 2 Help Fig 6 20 Create an Analysis Set Step 3 To add spots to this set click Add spots to set and click an appropriate 1 e housekeeping protein spot The cursor displays as a green cross The spot will be highlighted indicating that 1t has been added to the set If you want to remove a spot from the analysis set click Remove spots from set and click a highlighted spot Click Save The new analysis set is added to the list See section 7 1 for more information on creating analysis sets The total quantity of the valid spots in this analysis set will be displayed for each MatchSet gel in the table at the bottom of the dialog box 6 26 Analysis Tools The raw quantity of each spot in a gel will be divided by the total quantity of the spots in the analysis set in that gel according to the normalization formula Note The advantage of selecting multiple housekeeping proteins is that occasional minor
256. ew name and or version number for the new copy before the operation is performed Once this information has been entered the filtering operation will be performed 3 16 Invert Data If your image has light spots on a dark background 1 e the signal intensity of the background is greater than the signal intensity of the sample you will need to use the Invert Data function before you can analyze the image Note The Invert checkbox in the Transform dialog box section 3 12 b Other Transform Features inverts only the appearance of the image To invert your image data select Image gt Invert Data You may need to use the Transform function to adjust the appearance of your inverted image This command is reversible Select Invert Data again to revert to the original data 3 17 Text Overlays If you want to create and display textual notes directly on your image select Text Overlay Tools from the View gt Toolbars submenu This will open the Text Overlay Tools toolbar Text Overlay Tools x Select tool k ABC N r a M ef 3 Text tool Line tool Copy Paste Alignment tools Fig 3 24 Text Overlay Tools toolbar 3 38 Viewing and Editing Images Creating a Text Overlay To create a text overlay click the Text Tool then click on the image at ABE the spot where you want the text to appear This opens the Text Overlay Properties dialog box Text Overlay Properties Text color Bl z u bafa a
257. f the image on the camera CCD over a period of time The effect is analogous to exposing photographic film to light over a period of time Auto Expose Use Auto Expose if you want to take a single exposure but are uncertain of the optimal exposure time Note If you know the approximate exposure time you want 3 seconds you can skip this step and go directly to Manual Expose Click on the Auto Expose button to cancel Live Focus mode and begin an automatic exposure The Auto Expose button will appear selected throughout the exposure During the auto exposure the image is continuously integrated on the camera CCD until it reaches a certain percentage of saturated pixels This percentage is set in the Options dialog box Default 0 75 percent See C 5 Options Step IIl Acquire Image Click on Auto Expose Auto Manual Live button appears selected D Expose Expose Acquire Exposure Time sec SER Fig C 5 Selecting Auto Expose Can see Exposure Time automatically changing C 6 Appendix C ChemiDoc XRS Once an image has reached the specified percentage of saturated pixels it is captured and displayed in the video display window Auto Expose 1s automatically deactivated and the exposure time appears active in the Exposure Time field Note If you are having difficulty auto exposing your sample you can use Manual Expose to adjust your exposure time directly Most non chemiluminescent applications
258. fied and highlighted in each of the gels of the MatchSet To select a different analysis set click the pull down button next to the analysis set name and select from the list The number of spots in the set is displayed next to the name Integrated Excision Tool li Excision Gel Selection H5 Demo BF ie Step 1 Select Analysis Set Set name Guant Demo Spot count 23 Step 2 Set Selection Mode Auto select Tolerance ratio 2 50 a Gels selected 6 7 Single gel Include spot qualit Single group Bi ES Manual Table options Image options Multiple cut options U Show quality O Show ratio Show all gels Display cuts Enable multiple cuts spot Max cuts spot E Specify cuts gt Subo z00m or Cut Active gels only Step 3 Specify cute SSP Demo 30degree Diemo pena Diemo 30degree3 Demo Demo 9 1 b In this step you can select parameters for automatically selecting which gels to cut E 001 1304 PI Po P ho ro 1 on Es SS aAa a DI oo E ri Z Totals 355546 3 409533 1 2555080 O 1180016 5 376959 3 1482128 8 152949 2 1692068 9 311594 7 3204593 0 1467975 4 414358 7 224r 239 5 2600792 0 20r 6925 3 1204492 6 370299 7 2451871 3 1456325 6 ar 45 oF 35 El ea ENSERES JPA 2 El O Proceed af Close ES Cancel Ma Help Fig 9 3 Excision Gel selection Step 2 Step 2 Set
259. format ABnn A is the molecular weight grid letter B is the isoelectric point grid letter and nn is the number of the spot within that section of the grid Assigning DSNs After you have entered MrpI values for known spots you can create a DSN grid using this data by selecting Assign DSN Grid from the Analyze gt Spot Numbers submenu This automatically assigns a DSN number to each spot in the Master Qo C90 O Q manne Pra pap PAPGOT Fos fant D Fm IU 121 1820 LALA HE al mK LA LAOLEO1 e Ana I ued JA Ha dea HA21 dy phat Do c Hat GAO GHO1 pd Cc database mmber grid CEE TA E DSN pr FEF FDO FHO2 MS00002 2001 10 04 Madter Modifie srr ca it deal Modifie EAL aut Es EAU Eau L EHUA ate Plot DSN Grid Plot DSN Grid Counts Display DSNs Fig 6 25 DSN grid counts and numbers To display the grid this does not occur automatically when the grid is created click in the Master and select Plot DSN Grid A grid of vertical and horizontal axes will appear with the letter assignments of each axis displayed in the window margins To see the spot assignments select Display DSNs To read the numbers you will probably need to zoom in on a smaller region of image Transfer DSNs is used to copy the DSN numbers from one higher level MatchSet to another See section 5 8 b Higher Level MatchSets 6 35 PDQuest User Guide Transferring DSNs in a Higher level MatchSet Transfer DSNs is used t
260. g To add arrowheads to a line make sure the Line Tool or Select Tool is assigned to your mouse then double click the middle of the line A dialog box will pop up with options to add arrowheads to one or both ends of the line Moving and Copying Text Overlays and Lines You can move copy or delete a single overlay line or a group of overlays lines within an image You can also copy and paste between images First you must select the object s Click the Select Tool button on the Text ES Overlay toolbar To select a single overlay or line click 1t To select multiple objects either drag a box around them or hold down the SHIFT key while you click them one at a time When dragging to select a group of objects make sure that you completely surround all the objects to be selected Each selected overlay line will have a green border e To move the selected object s position your cursor over the selection and drag e To copy within an image hold down the CTRL key while dragging the selected object s The copy will be created and dragged to the new position e To delete the selected object s press the DELETE key EP eTo copy between images click the Copy to Clipboard button on the Text Overlay toolbar then open or select the image you want to copy to e Click the Paste from Clipboard button The copied object s will be pasted into the new image in the same relative position they were copied from 3 40 Viewing and Edi
261. g N 14 Custom Report Wizard The columns appear in the report in the order shown To change the order of the records click and drag the records in the list To remove a column highlight the column in the Records to Report field and click the arrow pointing to the Select Columns field Once you have selected your table and columns click the Record Report button This opens the Report for Table Table Name dialog From this dialog you can print the report or export it as a spreadsheet Use the arrows to scroll through the pages N 24 Appendix N WorksBase i Report For Table CONTAINER _TYPE CONTAINER_TYPE CONTAINER_UNITS_TOTAL Microtiter plate 96 MALDI plate 10x10 100 MALDI plate 12x8 96 fl im al daros ejej a Fig N 15 Custom Table Report There is no filtering option for this type of report However once you export the report to a spreadsheet it can be filtered in your spreadsheet application N 9 b Column Description Report This report presents the meta data in WorksBase in a user viewable form This report is very helpful for software developers N 25 PDQuest User Guide N 26 Index Numerics AI E EE E E ENE ese EAEE E EE A E ease 3 18 A ABEC OMiparisOm aaa gal a a a ae ue hs 11 7 PROMO PID ee E 2 43 PGS POL aaa a Seana edd a ae aa arenas 4 16 Add SPOL 0 Standard asin dit ilrtalia 5 28 Analysis Sel Mana is as 7 2 ARAS Si iones 7 12 7 15 7 17 7 20 SS A A O A tetas 7 1
262. g will stop This will prevent you from losing excision records and the associated Mass Spec identifications Tip If you want to re detect an existing image use the Crop and Copy command to make a copy of it Set the crop box to cover the entire image N 7 Protein Identification This involves spot excision digestion of the resulting gel plugs mass spectrometry to obtain a peptide signature or de novo sequence and searching against a reference database to obtain identifications for the proteins The results are imported into PDQuest annotation categories for easy retrieval N 7 a Excision Save MatchSet in WorksBase The MatchSet MUST be saved in WorksBase before performing excision All the preceeding steps acquisition automated detection and matching analysis set creation and gel selection may have been performed on disk However before performing the actual excision step the MatchSet must be saved in WorksBase using the Save in WorksBase command from the File menu Select MTP At the start of excision you will be asked to select create a microtiter plate record to receive the gel plugs You should use labelled microtiter plates to avoid confusion and enter the plate label at this step N 18 Appendix N WorksBase il Select Microtiter Plate E X Select plate to edit fi ha Plate name cF 20020130081825 Plate size lo First free well ADE Speciy starting well Current tun serial fi v OK X Ca
263. g parameters using the Processing Parameters pulldown list You can specify a mass spec run number in the MS Run Sequence Number field The format for use with Mascot check box allows you to include the MatchSet name in the sample identifier string so that results from the specific MatchSet can be filtered out of the master search log The purpose of this is to include all necessary information in the MSDataName tag in the file This tag eventually is used by Mascot as the title in the searches log file See your MassLynx documentation for processing your raw data for use with the Mascot search engine When you have made all your selections click Done 10 2 Importing MassLynx Results After you have completed a mass spec analysis and processed the data using MassLynx you can import the results back into PDQuest and include them in annotations for the spots you have analyzed Note Before importing MassLynx results you must select the Bio Rad output checkbox in the MassLynx project settings To access this checkbox go to the ProteinLynx menu in MassLynx and select Setup ProteinLynx One of the setup steps is called Select Output In this step under Database Searching select the Bio Rad full text output checkbox With the appropriate MatchSet open select Import MassLynx Results from the Identification menu In Step 1 the MatchSet name will appear in the MatchSet field Select the instrument used in the analysis by selecting the
264. g the extension select All files in the Files of type drop down list of the Open dialog With this checkbox selected when you save a scan a backup copy will be placed in the same directory as the scanned image Windows backup files will have an sbk extension Macintosh backup files will have the word backup after the file name Simple Acquisition Mode The Simple Acquisition mode option allows you to reconfigure the Gel Doc acquisition window to simple mode In simple mode the acquisition window contains the same steps as the advanced acquisition window with the exception of Step IV Optimize display and Step V Analysis Manual exposure is also disabled in simple mode O Live Focus IRIS ZOOM FOCUS 4 Open 4 Tele Near Y Close Y Wide Y Far Step Il Image Mode cuy White Step Ill Acquire Image Auto O Expose O Freeze Step IV Select Output video Pint E Save Opions E Hep Fig A 10 Simple Acquisition Window Appendix A Gel Doc To change the acquisition window to Simple Acquisition Mode check the box marked Simple Acquisition Mode The change will take effect the next time you open the acquistion window A 15 PDQuest User Guide A 16 Appendix B ChemiDoc Fig B 1 ChemiDoc Before you can begin acquiring images the ChemiDoc system must be properly installed and connected with the host computer See the ChemiDoc hardware manual for install
265. g to the selected replicate group PDQuest User Guide 1 Group Consensus Be x The Replicate Group Consensus Tool allows the user to use spot matching information within replicate groups to edit spot detection errors The Consensus auminary table shows the number of spots within a given replicate group that are matched across a given number of gels in this group Step 1 Define Acceptance Criteria for Presence of Spots Consensus Summary Spots matched acrass 1 Gel 2 Gels 3 Gels Ambiguous sox feje a z 144 ee Update Consensus Rematch Help Step 2 Review Individual Spots Active spot group 1 of 799 T P ra Quantity Graph Manual Automatic Qu En Auto Remove el Auto Cancel Global Actions Ae Auto Add All fl Auto Cancel All Auto Remove All ls Select Spot in Gel X Display 55Ps The left and right arrow keys may be used to change the active spot group SF Auto Add EX Close H Help Fig 6 11 Replicate Group Consensus At the top of the Group Consensus tool is the Consensus Summary listing all replicate groups in the MatchSet The columns represent the number of spots found on one gel two gels three gels and so on depending on the number of gels associated with the replicate group The number in the 1 Gel column represents the number of spots found on only one gel the number in the 2 Gels column represents the number of spots found
266. ges the ChemiDoc XRS system must be properly installed and connected with the host computer See the ChemiDoc XRS hardware manual for installation startup and operating instructions C 1 PDQuest User Guide To use the ChemiDoc XRS you will need to have the Bio Rad supplied acquisition board installed in your PC or Macintosh The drivers for this board will be installed when you install the main software application Make sure that your ChemiDoc XRS camera is turned on If the camera is not turned on the ChemiDoc XRS acquisition window will not open Simulation Mode Any of the imaging device acquisition windows can be opened in simulation mode In this mode an acquisition window will open and the controls will appear active but instead of capturing real images the window will create dummy images of manufactured data You do not need to be connected to an imaging device to open a simulated acquisition window This is useful for demonstration purposes or practice scans To enter simulation mode hold down the CTRL key and select the name of the device from the File menu The title of the acquisition window will indicate that it is simulated C 1 ChemiDoc XRS Acquisition Window To acquire images using the ChemiDoc XRS go to the File menu and select ChemiDoc XRS The acquisition window for the instrument will open displaying a control panel and a video display window C 2 Appendix C ChemiDoc XRS ChemiDo
267. ght All Pan Down Graph Retreat Graph Advance All Entire Images Grab All Same Hide All Overlays show All Crosshairs Quantity Table show Offsets Mark Unmatched Spots Find Spot Margin Graph Interchange All Images save All Control Configure Sub Windows com Out El Fig 2 5 Keyboard Layout The pulldown menus also list the shortcut keys for the menu commands Note Mouse assignable commands behave differently 1f you assign them using the keyboard versus selecting them from the menus or toolbars For example to use the Zoom Box command as a keyboard command position your cursor on the image where you want to begin to create the magnifying box then press the F2 key The command is assigned to your mouse and immediately activated so that when you move your cursor over the image the zoom box is created When you General Operation click the mouse button once the defined region is magnified and the tool is automatically deassigned from your mouse 2 3 File Commands and Functions This section describes the basic file commands and functions of PDQuest Note Print and Export commands are described in Chapter 12 2 3 a File Types in PDQuest The types of files supported by PDQuest are Raw 2 D scans gsc Filtered images gim Gaussian images gsp Calstrip scans csc MatchSets ms Backup scans sbk Backups cannot be opened in Windows you must change the extension to gsc using Window
268. gin to appear in the scanning window line by line To interrupt a scan click on the Stop button A message will ask you to confirm the interrupt and then you will be asked if you want to keep the partial scan This feature is useful if you overestimated the size of the area you selected After the scan is complete a window will open displaying the scan image at which point you can analyze and save it Note The image will open with a default file name that includes the date time and if applicable user name However unless you have selected Auto Save After Scan the file will not be saved until you select Save or Save As from the File menu D 9 PDQuest User Guide D 6 Calibration If you have installed a calibration overlay you can automatically calibrate your transmissive and reflective scans Calibration overlays for the GS 700 can be ordered from Bio Rad To set the automatic calibration settings click on the More Options button in the GS 700 acquisition window This will open the Densitometer Options dialog box Densitometer Options Calibration E Calibration On On the GS 00 this requires that the calibration overlay be installed Calibrate Before Every Scan Recalibration la The Densitometer will be re calibrated Interval minutes after this time has expired E A calibration report will be printed each time the Lj Calibration Report Densitometer is calibrated Calibrate Mow Edit Step Tablet
269. gnment has been completed set the confidence threshold to an acceptable level We recommend it to be a high value The spots that meet this level display in green The spots below that confidence level display in red Step 4 Alignment Confirmation x Display cropped image Be Jump and Confirm Confidence at Spot 72 Confidence Threshold Cuts below confidence 0 Total cuts 2 1m ro Manual Correction drag any cut mark on camera image anytime Fig 9 12 Alignment Confirmation Use the zoom box button to magnify the images for greater visibility LS Jump and Confirm Jump and Confimn Click the Jump and Confirm button to jump to a view of the next spot in the cut list whose alignment is below the specified confidence threshold The spot being confirmed will be displayed in blue and will be at the center of the window If the spot is aligned correctly in the two images click on the Jump and Confirm button again to jump to the next spot below the confidence threshold If the spot is not correctly aligned drag it on the 9 26 Integrated Excision Tool camera image to the correct position Note that you can in fact drag any spot on the camera image any time to manually correct its alignment Repeat the above procedure for every spot to be cut in this gel D Align Anahi ard Cobras sampled y Famed Cutter Comes Image Matched o o spots i Xy b b E sa _ gt jx g Spot displaye
270. h the stage edges Click Ready to proceed You may also skip this step return to the previous dialog cancel the action or ask for help Step 3 The fourth dialog prompts you to place the coomassie stained PVDF membrane on the mat Click Yes or No At any point in the procedure you can move back to repeat a step Note Calibration creates several parameter and image files in the Install prm directory in the Discovery Series directory on your hard drive These files should not be moved deleted or altered or calibration will be affected 8 8 Basic Excision Tool When calibration is complete you can proceed with normal use of the spot cutter Once the cutter is calibrated you do not need to repeat the procedure during normal day to day use You must recalibrate if e The instrument is moved e You install or remove the lens filter after calibration 8 2 Cutting Spots After you have focused the camera and calibrated the spot cutter you are ready to begin cutting spots The Basic Excision tool control panel is designed to guide you through the procedure 1 Acquire Image In this step you capture an image of your gel or membrane using the spot cutter camera 2 Specify Cuts In this step you identify the spots in the image that you want to cut 3 Select the Cut Run Options These options can be adjusted for each cut run 4 Set Wash Options Select the wash stations to use after each cut by clicking on one or more checkbo
271. h gels but was missed during matching To create a Qualitative Analysis Set select Qualitative from the Select Set Type dialog Analysis Sets and Annotations Ll Analysis Set Template Matchset HalfStock a es Xx Name Description Options x Mark spots ye _ Estimate missing spots JEstimate saturated spots Display selected sets Mark spots on e Master gel Operand gels All gels f Repl Gels B Han Classes Show 2 Window labels _ 55F number Method C OninB Format Min fold over background 10 0 Onind Spot court 173 wf Save X Cancel z Help Fig 7 3 Qualitative Analysis Set dialog Enter a name and description of the analysis set Note If you have Normalization enabled normalization parameters will be used for spot quantities See Section 6 7 Normalization for further information Qualitative Analysis Set Options The following options help refine the analysis set Estimate missing spots If this box is left unchecked then the spot must be detected in 100 of the gels in the group for it to be included in the analysis If the box is checked the spot 7 7 PDQuest User Guide must be detected in gt 50 of the gels in the group to be included This is important in qualitative analysis because the analysis is based on whether spots appear in one group or class and do not appear in another Estimate saturated spots This option estimat
272. h green letters e Manually matched spots are labeled with purple boxes e Unmatched spots are circled in red These are also displayed using the Mark Unmatched Spots command Shift F8 on the Match gt Edit Matches submenu e Landmarks are labeled with green triangles These are also displayed using the 5 21 PDQuest User Guide Mark Landmarks command F8 on the Match menu and toolbar e Spots added to the Master are labeled with blue boxes These are also displayed using the Mark Added Spots command AIt F8 on the Match menu and toolbar menu or toolbar and click on a spot in the Master or use the F9 key command on the spot The spot will appear highlighted with a yellow box in all the gels in which it has been matched In gels where it is unmatched a circle will appear where the spot would be located 5 5 6 Show Vector Offsets Vector offsets are lines showing the distance and direction between the spot centers in the member gels and the corresponding spot centers in the Master They provide a visual tool for judging gel alignment and distortion and the accuracy of matching a To highlight individual matched spots select Show Match from the Match Select Show Offsets from the Match menu or toolbar and click in a T subwindow to display the offset lines or use the Shift F7 key command in the subwindow Look for the following conditions that indicate possible matching problems e Offset lines that cross each other this is a ke
273. han percent of the pixels in your image saturated Consequently the default value for this field is 0 75 percent Appendix C ChemiDoc XRS C 6 Other Features Display Options Highlight Saturated Pixels Image file size 2 60 Mb Options Help Fig C 13 Other ChemiDoc XRS acquisition window features Highlight Saturated Pixels When this box is checked any saturated pixels in the image will appear highlighted in red in the scan window and in the pop up image window To view hide saturated pixels in the pop up image window use the Image gt Transform command File Size of Images Image File Size shows the size of the image file you are about to create This size is determined by the bin setting used for the selected application If you do not have enough computer memory for the specified file size an error message will appear when you attempt to acquire an image Macintosh users can increase the application memory partition See your Macintosh computer documentation for guidance PDQuest User Guide Appendix D GS 700 Imaging Densitometer Fig D 1 GS 700 Imaging Densitometer Before you can begin scanning images with the GS 700 Imaging Densitometer your instrument must be properly installed and connected with the host computer See the hardware manual for installation startup and operating instructions PC Only A Note About SCSI Cards The GS 700 is connected to your computer by a Small Computer System I
274. have a SCSI card installed in your PC If you have a PC with a Windows 98 or Windows ME operating system you may also need to load the SCSI and WinASPI drivers that came with the card Simulation Mode Any of the imaging device acquisition windows can be opened in simulation mode In this mode an acquisition window will open and the controls will appear active but instead of capturing real images the window will create dummy images of manufactured data You do not need to be connected to an imaging device to open a simulated acquisition window This is useful for demonstration purposes or practice scans To enter simulation mode hold down the CTRL key and select the name of the device from the File menu The title of the acquisition window will indicate that it is simulated H 1 Fluor S MAX Acquisition Window To acquire images using the Fluor S MAX go to the File menu and select Fluor S MAX The acquisition window for the imager will open displaying a control panel and an image display window H 2 Appendix H Fluor S MAX E Fluor S MAX Multilmager E Step Select Application Select Coomassie Blue User White Trans HiSens Scan Dimension 80mm 160mm 250mm 300mm Step II Position Focus Position Focus x Show Alignment Grid Step Ill Set Exposure Time sec Eo tl O Preview Q Acquire Olive Acquire 4s Stop Display Options Highl
275. he acquisition of a new Flat Fielding reference image Acquire Click on the Acquire button to capture a single image for each enabled channel An exposure will be taken for each enabled channel based on the time selected for that channel in Step III The progress of each exposure will be displayed in the Exposure Status bar at the bottom of the acquisition window K 11 PDQuest User Guide Exposure Status A JAcquiring Image Fig K 6 Exposure Status bar when acquiring an image Depending on which dark subtraction type you have selected under Options see section K 6 Options a dark image may be acquired immediately following image acquisition If you want to stop an acquisition that is in progress click on the Stop button The current acquisition will be terminated If multiple channels are selected you must click on the Stop button once per channel to stop all acquisitions After an image has been acquired a separate window will pop up containing the new image You can then analyze the image using the analysis functions Optimize Exposure Optimize Exposure allows you to specify an interval over which a series of progressively longer exposures are taken All exposures are then displayed on the screen and you can choose the one that provides the best image Note Multiple exposures will be taken for only the selected multichannel tab Other channels even if enabled will not be used Illumination Flat Fielding wi
276. he action Performing a global action causes the consensus summary table to automatically update There is no need to manually update the Consensus Summary table 6 6 Sample Database The Sample database of PDQuest allows you to track gel sample associations With the sample database you can attach attributes to samples This is important for tracking two different gels of the same sample type For instance your MatchSet may contain a series of E coli samples but with different treatments such as temperature 6 6 a Associating Sample to Gels To associate samples to gels select Analyze gt Describe Samples gt Enter Sample Attributes 6 17 PDQuest User Guide ii Enter Sample Attributes Si z x Image Sample Treated Dilution A Done Sample 1 Yes 2 to1a stock2 Sample 2 No None amp View Report stock3 Sample 3 Ho 2 fold Import alfi Sample 4 Yes None Sample 5 Yes 2 to1a Sample 6 No None Pattee ss Add Sample Edit Attribute Fig 6 13 Enter Sample Attributes 4l Help With the Enter Sample Attribute window open click the cell under the Sample heading that corresponds to the gel for which you wish to add a sample then click Add Sample This opens a list of samples from which to choose Highlight the sample you wish to apply and click Done Ll Select Analysis Sample xj Sample Samplet sample1 sample2 sample3 sampled ff Done 1 New
277. he exact format necessary to complete the URL link to the page in the database for the specific protein information you are referencing If you are entering an annotation in a file name category the entry should be the exact file name you want to link to the spot If you are entering an annotation in the protname category the entry should be the protein name you want to associate with the spot To apply an annotation entry click the Apply Entry button The entry will be automatically applied if you select another spot or category to annotate To delete an annotation entry select the entry text and press the Backspace or Delete key Then click Apply Entry or select another spot to apply the deletion 7 24 Importing Annotations The Import Annotations tool allows you to import annotations from a text file This is useful for adding a large number of the same annotations to multiple MatchSets The import annotations tool is fairly flexible with the exception that the first column must contain the SSP numbers To import annotations click Analyze gt More Annot Tools gt Import Annotations This opens the Import Annotations dialog 7 28 Analysis Sets and Annotations Ll Import Annotations Matt 2002 03 18 Ee x File to import ltest tab tt Ed Header line SSPOcolorOsizeOO First data line ji 502 0blue Dsmalltish 00 x First line ts header X Confirm overwrites Column Delimiter f Comma e Tab Select Category antibod Add
278. he maximum aperture This reduces the depth of field allowing you to PDQuest User Guide more accurately focus the camera Then after focusing increase the f stop to the desired setting After you have positioned your sample click on the Focus button and look at the image in the acquisition window while aligning the two focusing arrows on the camera lens While focusing the camera will limit its focus to a small portion of the sample this will not affect any zoom lens adjustments you may have made When you are finished focusing click on the Stop button H 4 Step Ill Set Exposure Time When you are ready to capture an image you will need to select an exposure time Exposure refers to the integration of image captures on the CCD over a set period of time The effect is analogous to exposing photographic film to light The exposure time you select should be based on your application and your best guess as to what exposure will give you the best image Note The minimum exposure time in trans illuminated mode is 1 second The minimum exposure time in epi illuminated mode is 0 1 second You can enter an exposure time in seconds directly in the field or use the Arrow buttons to adjust the exposure time in 10 percent increments Step lll Set Exposure Time sec t y D Preview Fig H 8 Selecting an exposure time The following table provides recommended exposure times for various applications H 8 Ap
279. he recut request dialog then click Begin Resume to perform the recuts Re cuts in Basic Excision Tool The specification for the ProteomeWorks spot cutter spot pick up is greater than 95 effective at picking up a spot that has been cut from a gel There may be some spots that have not been picked up and these can be re cut The efficiency of pick up on the second cut is again gt 95 so the spot is reliably picked up the second time The missed pick ups are detected by the user looking in the microtiter plate for empty wells However with fluorescent gels the clear gel spot with no visible stain is very difficult to see in the microtiter plate wells In order to assess the wells of the microtiter plate the re cut message on the screen for the fluorescent light source will differ from the white light message Use the following procedure for re cutting spots in fluorescent mode When UV Fluorescent imaging has been used on the spot cutter at the end of the cut run the software will prompt with Confirm cuts 8 19 PDQuest User Guide a POQUEST Ea Confirm cuts Acquire an image of the MTP in UY mode to check whether re cuts are needed es Mo Fig 8 14 Confirm cuts window Step 1 Answer Yes to bring up the following on screen instructions for imaging the microtiter plate in UV mode fh Image Microtiter Plate Step 1 Setup MTP AN Do not move gel on stage Cover gel with Gel Cutting Sheet P
280. her you want to 1 crop the original image 2 save a copy of the area inside the crop box as a separate image keeping the original image intact or 3 cancel out of the cropping operation 3 27 PDQuest User Guide q 7 50 33 Kb 259 x 199 pixels 77 7 x 35 9 mm i POQUEST x Ready to crop T8237 ve y4 20 Scan Crop current image or crop copy of image Crop Cancel Fig 3 18 Crop box and pop up Crop dialog If you select Crop your cropped image will be displayed in the image window If you select the Copy and Crop button a dialog box will open in which you can enter the name of the cropped image its version number exposure number and or file type Click OK and the image window will display both the original image and the cropped image in subwindows 3 13 a Advanced Crop Commands To ensure that your scans are exactly the same size and shape you can use the tools on the Image gt Advanced Crop submenu to save the crop box from one scan and apply it to others These tools also allow you to crop a gel and calstrip out of the same scan To define and save a crop box and apply it to another scan 1 Select Define Crop Area from the submenu and drag a crop box on an image Position the box as described in Cropping Images 2 Select Place Crosshair from the submenu and click a landmark inside the box that is present in all the gels you want to crop This could be a spot or some other 3 28 Viewi
281. here you are more interested in resolving in the y dimension to determine the size of bands and the spacing between them E 8 Appendix E GS 710 Specifying Your Own Resolution If you select Oversample under More Options you can specify your own resolution within the range of 43 169 microns micrometers With Oversample selected enter values directly in the fields next to X resolution and Y resolution in the main acquisition window Step lll Select Resolution X resolution so 0 Microns Select Y resolution so ol Micron Image file size 0 10 ME Fig E 8 Entering a custom resolution with Oversample selected Image File Size Image File Size under Select Resolution shows the size of the scan file you are about to create If you do not have enough computer memory for the specified file size an error message will appear when you attempt to scan If this happens select a lower resolution or decrease the size of the area to be scanned Macintosh users can also increase the application memory partition See your Macintosh computer documentation for guidance E 5 Calibration Images scanned using the GS 710 are automatically calibrated The scanner plate has a built in calibration overlay for both transmissive and reflective scanning e The transmissive calibration strip calibrates the densitometer from 0 to 3 0 O D e The reflective calibration strip calibrates the densitometer to 2 0 O D The first time
282. hers This makes it easier to correlate data from the member MatchSets With the higher level MatchSet open select Transfer SSPs from the submenu In the first pop up box select the MatchSet member you want to copy from in the second box select the member you want to copy to You will be asked to confirm the transfer When the transfer is complete the number of SSP numbers transferred will be listed in an alert box Repeat the procedure to apply the numbers to all the members in the MatchSet 6 10 Database Spot Numbers Database spot numbers DSNs are an alternative to the SSP numbers The DSN system provides a more flexible way to number protein spots in a Master in that 1t indicates the general location of each spot A coordinate grid can be established for each axis based on molecular weight and isoelectric point The grid coordinates are identified with letters A R excluding I and O because they look like numbers The letters are intended to represent specific MrpI ranges so that the numbering between different gels can be comparable These ranges are as follows Mr values 0 10 A 87 117 J 11 14 B 118 158 K 15 19 C 159 215 L 20 25 D 216 293 M 26 34 E 294 398 N 35 46 F 399 541 P Mr values cont 47 63 G 542 736 Q 64 86 H 737 1000 R pI values 0 2 A 7 0 7 5 J 2 3 B 7 5 8 0 K 6 34 Analysis Tools 3 4 C 8 0 8 5 L 4 5 D 8 5 9 0 M 5 0 5 5 E 9 10 N 5 5 6 0 F 10 11 P 6 0 6 5 G 11 12 0 6 5 7 0 H 12 14 R DSNs have the
283. high level MatchSets PDQuest can be used to simultaneously analyze thousands of spots on hundreds of gels Data can be exported to other applications such as spreadsheets for further analysis 1 1 PDQuest User Guide Mass Spec Analysis PDQuest is part of Bio Rad s ProteomeWorks protein analysis package and controls Bio Rad s ProteomeWorks Spot Cutter You can cut spots from gels or membranes using PDQuest digest them and perform advanced protein analysis using MicroMass s mass spectrometry instruments and software Data from MicroMass can then be imported back into PDQuest to be included in spot annotations And More Scan files acquired in PDQuest can be analyzed using other Bio Rad Discovery Series software applications such as Quantity One or Diversity Database Scans can be converted into TIFF format for easy compatibility with other applications 1 2 Digital Data and Signal Intensity The Bio Rad imaging devices supported by PDQuest are light and or radiation detectors that convert signals from biological samples into digital data PDQuest then displays the digital data on your computer screen in the form of gray scale or color images A data object as displayed on the computer is composed of tiny individual screen pixels Each pixel has an X and Y coordinate and a value Z The X and Y coordinates are the pixel s horizontal and vertical positions on the image and the Z value is the signal intensity of the pixel
284. his scaling factor is used in to make the numbers in the formula more manageable O is the standard deviation of the Gaussian distribution of the spot in the direction of the x axis and is the standard deviation in the direction of the y axis These are measured in Us Note that this formula results in units of OD IU for images measured in ODs and units of counts for images measured in counts 4 24 Note Detecting and Editing Spots This formula results in more accurate quantitation of imaged spots than summing the intensities of the pixels in the spot Measuring the area under the Gaussian model compensates for peak intensity values that approach image saturation as well as background intensity that submerges the intensity of a spot at its edges To display the quantities of the spots in a single image use Show Quantity AIt F6 Spots menu in the image window To display the quantities of all the spots in all the subwindows of a selected window select Show All Quantity from the Spots menu Low Quantity Spots You can define a set of spots that do not meet a threshold quantity level From the Spots gt Low Quantity Spots submenu select Define Low Quan Set then click in the MatchSet or scanset Gaussian image In the pop up box enter a threshold quantity value All spots below this threshold will be flagged as low quantity When you click OK a pop up box will list the number of low quantity spots in the image and
285. http www expasy ch cgi bin sprot search de s When you are finished creating the category click Done When you create an annotation entry for a spot in this category it should be the exact format necessary to complete the URL link to the page in the database for the specific protein information you are referencing To open a URL link right click on the spot and select Browse Annotations The HTML page for the spot will open and the URL category name will be displayed as a link Click on this link to access the protein information in the database or Web site 7 25 PDQuest User Guide File Name Categories This type of category provides a link between spots and files on the user s computer network or removable disk drive For example you can open a Protein Probe file linked to a spot from within PDQuest To create a file name category open the Add Category dialog box enter a name for the category e g Protein Probe then select the Filename option button In the File Extension field type the extension of the file type you want to link e g Protein Probe files have the extension prp Use the Browse button next to the Files in Folder field to select the directory where the files in this category located il Edit Category CS x Category Mame assoprot Description Associated Proteins Type of entry e Text A Ke ANC rd f URL arg Filename wf Done X Cancel Help Fig 7 12 Editing a text
286. ice and capture the image using the controls Gel images are saved on your hard disk network file server or removable storage media The displayed image in PDQuest is ready for analysis Image Sizing and Orientation In this step you adjust the size and orientation of the image by using the cropping and rotating tools on the Image menu Introduction Spot Identification Now you are ready to identify the spots in the gel The Spot Detection Wizard automates the process of selecting the proper spot detection parameters for your gels Using the Wizard you select the parameters study the results of spot detection then adjust the parameters until you have identified all spots of interest in your gels When spots are detected in PDQuest the original gel image is filtered and smoothed to clarify the spots then three dimensional Gaussian spots are created from the clarified spots The end result is three separate images the original unaltered scan 2 D Scan the filtered and processed scan Filtered image and a synthetic image containing the Gaussian spots Gaussian image Filtered Image Gaussian Image Fig 1 3 Images created during spot detection All spot matching and analysis in PDQuest are performed on Gaussian spots What Are Gaussian Spots Fuzzy streaked or overlapping spots in a 2 D gel can be difficult to accurately distinguish and quantify Because the image profile of an ideal spot confirms to a Gaussi
287. ick Delete 11 2 Histogram Graphs Histogram graphs can be used to quickly compare a spot s quantity in each member of a MatchSet A brief look at a histogram can give you a sense of the general trends in spot quantity Histograms are also useful as tools for detecting problems such as spots that are not matched and spots whose quantitation is suspiciously off the average Reviewing the histograms of spots in an Analysis Set is a quick and easy way to confirm that the members of the Analysis Set meet the criteria that was specified when the set was created 411 13 Un norm Un norm SSP 0111 SSP 0112 SSP 0113 SSP 0114 10 230 Un norm Un norm SSP 0116 SSP 0117 SSFP 0116 sor 0115 Fig 11 5 Sample Histogram graph Graphs and Reports 11 2 a Data Displayed in the Histogram Each bar in a histogram for a spot represents the spot s quantity in a member of a MatchSet If replicate group quantitation is selected each bar represents the spot s quantity in a replicate group The bars from left to right represent the gels in the order in which they were loaded As long as the spot is matched to the Master you can display a histogram for it Maximum Quantity a Normalization Units SP 4203 Fig 11 6 Sample histogram The Standard Spot SSP number is displayed beneath the histogram The number at the upper right of the histogram is the quantitation of the maximum bar in the graph The other bars are drawn proporti
288. ick the Live Focus button and frames will be captured and displayed at about 10 frames per second depending on the speed of your computer You can use live mode to zoom focus and adjust the aperture on the camera while positioning the sample within the area Note ChemiDoc XRS features a motorized zoom lens that can be controlled directly from the acquisition window using the Iris Zoom and Focus arrow buttons Click on the Up Down buttons while viewing your sample in the window to adjust the lens Step l Position Gel D Liver Focus IRIS 00M FOCUS e 4 el 32 Show Alignment Grid Fig C 4 Live Focus button and camera control buttons You can also select the Show Alignment Grid checkbox to facilitate positioning Note After positioning your sample you should check the Imaging Area dimensions under Options see C 5 Options to make sure that they conform to the size of the area you are focusing on To determine the size of the area you are focusing on you can place a ruler in the ChemiDoc XRS hood so that it is visible in the image C 5 PDQuest User Guide C 4 Step Ill Acquire Image The ChemiDoc XRS control panel has several features for creating image exposures You can take an automatic exposure based on the number of saturated pixels in the image you can enter a specific exposure time or you can take a series of exposures and select the best one Note Exposure refers to the integration o
289. ig N 1 WorksBase submenu WorksBase supports e Lab image data analysis software e Lab data from proteomics Mass Spec instruments e Bioinformatics queries and data analysis tools PDQuest User Guide N 1 Features WorksBase features a data warehouse that integrates your data with reference data to e incorporate reference database annotations into research results e include your own additions to reference data You can use WorksBase s query and reporting tools for sample identification and tracking protein identification expression analysis and bioinformatics queries WorksBase s user interfaces describe sample sources and experimental protocols The query tools correlate protein sequence identities that are assigned by Mass Spec with changes in expression level Changes in expression levels range across sample variables disease states and treatment conditions You can query annotations from reference databases to discover patterns among proteins N 2 Getting Started with WorksBase N 2 a Overview WorksBase Software for Proteomics provides direct access to the records and tables in your database Use the Proteomics tools to track your experiments and projects Tracking ranges from sample preparation through managing information about projects experiments protocols gels samples containers and results You can add records explore tables and records in the database and perform queries N 2 b Installation WorksB
290. ight Saturated Pixels Image file size 0 50 Mb Options 7 Help Exposure Status Temperature 20 0 Fig H 2 Fluor S MAX acquisition window When the Fluor S MAX window first opens no image will be displayed The control panel has been arranged from top to bottom to guide you through the acquisition procedure There are four basic steps to acquiring an image using the Fluor S MAX 1 Select the application 2 Position and focus the gel or other object to be imaged 3 Set the exposure time 4 Acquire the image H 3 PDQuest User Guide H 2 Step Select Application To set the appropriate filter and other parameters for the type of object you are imaging click on the Select button under Select Application The available applications and their associated settings are listed in a tree that expands from left to right Mucleic Acid Gels Blotting Copper Stain Densitometry Silver Stain Custom SYPRO Orange SYPRO Red Zinc Stain Coomassie Blue Clear White Trans Fig H 3 The application tree in the Fluor S MAX acquisition window First select your general application then select the particular stain or medium you are using If you select the chemiluminescent application under Blotting you also have the option of selecting High Sensitivity or Ultra Sensitivity see below When you select an application the software automatically sets the appropriate standard filter S20LP 530DF60 61
291. ill appear on the graph All spots between these lines have quantities in the two items that fall within the selected fold factor range The correlation coefficient for the regression line is displayed on the graph A coefficient of 1 00 indicates that the two items are perfectly similar while a low coefficient value e g 0 40 indicates that the two are not very similar The number of spots that appear in both is also listed on the graph If you click a point in the graph that spot will be highlighted and magnified in the actual MatchSet To print the currently displayed scatter plot click the Print button in the window To print scatter plots comparing every gel in the MatchSet with every other gel click the Print All button This opens the Scatter Plots Report dialog Enter a title researcher and institution and click Done This opens the report viewer The report viewer allows you the option of printing or reformatting the report Click reformat report button to return to the Scatter Plots Report dialog 6 4 Replicate Groups Since most experiments are carried out in duplicate data analysis can be performed on each gel individually or on sets of duplicate gels grouped together The Replicate Groups function allows you to group sets of duplicate gels and determine the average quantities of their protein spots Note All duplicate gels must be in the same MatchSet before you create a replicate group To make a replicate group
292. ill open in live mode giving you a live video display of your sample If no image is visible make sure the camera is on check the cable connections make sure the iris on the camera is not closed and make sure that the protective cap is off the camera lens Also check to see that the transilluminator is on and working The control panel has been arranged from top to bottom to guide you through the acquisition procedure There are six basic steps to acquiring an image using the Gel Doc 1 Position and focus the gel or other object to be imaged 2 Select Mode 3 Acquire the image A 3 PDQuest User Guide 4 Optimize the display 5 Analysis 6 Select the output A 2 Step Position Gel The Gel Doc window will open in live mode giving you a live video display of your sample In this mode the Live Focus button will appear selected and frames will be captured and displayed at about 10 frames per second depending on the speed of your computer You can use live mode to zoom focus and adjust the aperture on the camera while positioning the sample within the area Note Newer versions of the Gel Doc feature a motorized zoom lens that can be controlled directly from the acquisition window using the Iris Zoom and Focus arrow buttons Click on the Up Down buttons while viewing your sample in the window to adjust the lens These buttons will not be visible if you are connected to older versions of the Gel Doc
293. image are listed at the bottom of each subwindow 5 3 Matching Spots After you have created a MatchSet and selected the Master template you are ready to begin matching spots and adding unique spots in the gels to the Master In a MatchSet spot data from the member gels are included in the Master via matching All the significant spots in your MatchSet should be matched with spots in the Master There are two types of automated matching Automated Matching and Classic Matching Matchsets Manual Match anual Unmatch Mark Landmarks Match Tools 4 4 AE PALA g Landry Match Analysis Identification New MakchSet MatchSet Tool Automated Matefing Landmark Unlandmark Classic Match All Gels Classic Match Gel Classic Match All awe p Show Symbols Show Symbols Show Offsets Laat ese Mark Landmarks Show Match Show Match FE Combine Overlapping Gels CyberGel ma Auto 4dd Spots to Master Add Spot to Master____ Add Spot to Master Mark Added Spots BE FF Remove Added Spot Fig 5 7 Match Menu and toolbar Using landmarks PDQuest automatically matches most of the spots in the MatchSet You then review the remaining unmatched spots in the member gels and either manually match them with existing spots in the Master cancel them or add them as new spots to the Master 5 11 PDQuest User Guide Each matched spot is marked with a green letter in the Master and the matche
294. imes recommended tilda K 9 al do e e ad ea a TEATE C 5 K 6 Mumnmaton Platillos K 10 MA NG AAA A ero O EE A K 17 Opm ZE ePOS iaa a E idos K 12 a AA N odesedaavouwaceoee C 9 OPINAS iria K 14 A E oenentaacnimethoeenaes K 7 A aie ca suetiakna canta naan ees a A K 10 Saturated pixels MiMi OMS seren sdustinlon palo K 18 SAVE OPON S ce avss senators e nce artatehearsaat tt atari eceeneave atancassmeta mance K 17 Select AN APP IC AMON reresign a E EA NOR K 3 Video IN TNs iden biopsia 2 24 View Patito SC aia ora 3 6 Viewing images Centeno On the CUISOF aro o a EE 3 9 Undoing view commands sud ta 3 4 3 7 Windows AsSienimno interchangine Images ci 3 3 E ZOOM ea aid 3 7 SUG OWS leida 2 40 DI a a ELE ESC GO ous 3 4 Windows and subwindows overview s ssssessesseseseesessesensessesessesensessrsensenersenese 3 1 Windows Mle dlocahions irena O 1 9 Works Base urea a aa a a a a RR N 1 Works Base Custont REDO S nit dad N 23 Index 19 PDQuest User Guide Z ZOOM BOX nerone rT a EE A AA ENS 3 5 Zoom AN ZOOM DUE odia aia 3 6 Zoom tools EChansino Denda 2 39 Index 20 The Discovery Series Image Analysis Software Software License Agreement READ THIS THIS IS A LEGAL AGREEMENT BETWEEN YOU THE CUSTOMER AND BIO RAD LABORATORIES INC BIO RAD CONCERNING THE ENCLOSED COMPUTER PROGRAM THE PROGRAM YOU SHOULD REVIEW THE FOLLOWING TERMS AND CONDITIONS OF THIS AGREEMENT CAREFULLY BEFORE OPENING THIS PACKAGE BY OPEN
295. in WorksBase Step 1 Select Gels To Analyze Use the Add button to select gels to be analyzed and matched You do not have to open the images before processing them The Add button allows you to select images from WorksBase whether or not they are open The Remove button removes a gel from the list The Master button designates the selected gel as the MatchSet master image This gel will be used as the starting point for constructing the eventual Master image As indicated on the dialog you can set the order of the members by dragging them in the list This order will be used when displaying the images as well as plotting bar graphs Appendix N WorksBase Step 2 Detect Spots on Gels This will automatically process all the selected images The spot detection parameters are stored in WorksBase and any parameters previously saved in the current dataset are available If you cannot use an existing parameter set press the Edit button to launch the Spot Detection Wizard and create a new parameter set The new parameter set will automatically be stored in the database and available from any other system connected to WorksBase Step 3 Create MatchSet Enter a name for the new MatchSet or click Auto to automatically create a name Enter a description and any comments regarding the MatchSet To create a MatchSet an experiment must be specified If the Programs are required or Projects are required options are checked in the Work
296. in the same well If you wish to add or remove cuts click on the cells to add or remove them Note that this will automatically switch the selection mode to Manual To return to the Single group mode click the option again A dialog opens asking you to confirm overwriting your manual edits Manual To choose gels and spots manually click the Manual option button The spots to be cut are selected in the table based on the prior selection mode You can then click on spots to add or remove cut requests To select all the cuts in all the gels click the Select all button Multiple gel cuts of the same spot are placed in the same well To clear the table of all the currently selected cuts click the Deselect all button Table Options The table in the Excision Gel Selection tool lists the spots in the analysis set by Standard Spot Number and their quantities in the different gels The columns are gels the rows are spots You can use this table to make cut requests or modify the auto selection of cuts Under Table Options click Show Quality to display the spot quality in the table cells in parentheses following the spot quantity Click Show Ratio to display a column in the table showing the quantity tolerance ratio across all gels for each spot This value is used in conjunction with the Tolerance Ratio setting described above 9 7 PDQuest User Guide Show All Gels will show columns for all the gels in the MatchSet If this is unse
297. ing When this checkbox is selected the software will warn you to move the filter slider either to chemi or the UV White position C 5 c Dark Subtraction At the time of installation a reference dark image file was generated and saved to your hard drive The purpose of this file is to reduce dark current noise generated from the CCD Dark current noise is typical of all CCDs and is a result of the accumulation of charge in the absence of light The default time of the reference dark image is ten minutes The default reference file is optimized for ChemiDoc XRS applications If you select to change the reference dark image ensure that the time of the reference dark image is of equal or greater duration than your typical sample image When the sample image time is longer than the reference dark image time the ability to detect very faint samples may be compromised To reset the reference dark image time select options in the ChemiDoc XRS acquisition window Select Reset Referenced Quantity One x Reset Reference Dark Image Options Dark Exposure Time sec eon Cancel OF Fig C 12 Reset Reference Dark pop up box Enter the desired time in seconds Select OK You will be prompted to close and re open the acquisition window Place the lens cap on the camera and close the door of the hood See ChemiDoc XRS instruction manual Select OK at the prompt and new reference dark images of the entered time interval will be
298. io Rad Radioisotopes K Screen Kodak Fuji Screen Chemi Screen Bio Rad Chemifluorescence ECL Plus Attophos Ethidium Bromide DNA Stain Gel Sybr Green amp II Sybr Gold J 6 Appendix J FX Standard FX Applications Sypro Orange Sypro Red Protein Stain Gel Nile Red Sypro Ruby Alexa 488 Alexa 532 Alexa 546 FITC Fluorophores FAM CY3 HEX R6G Texas Red DNA Sybr Green Protein Nano Orange a ssDNA Oligreen Microtiter Plate DNA Picogreen B Gal FDG GUS FDG Coomassie Blue Gel Blot Copper Stain Gel Blot Densitometry Silver Stain Gel Blot X Ray Film Grey Type Not supported in the FX Pro and FX Pro Plus systems First select your general application next select the particular stain or medium you are using and finally if appropriate select the intensity of your samples Note Some applications require an external laser If you choose one of these without having an external laser attached you will receive a warning To exit the tree without selecting press the ESC key Your selection and settings will be displayed below the Select button J 7 PDQuest User Guide Sample Intensity Many FX applications require that you select a sample intensity High Medium or Low from the application tree This is simply a rough estimate of how much sample is visible in your gel or other object If you are unsure of the level of intensity of your sample you can always select a
299. ion in spot size and intensity between gels that is not due to differential protein expression This variation can be caused by a number of factors including e Inconsistent cell numbers cell densities in the sample prep e Inconsistent sample prep efficiency due to variations in reagents protocols etc e Handling errors resulting in some sample loss during prep e Pipetting errors during sample prep e Sample loss during gel loading e Inconsistent staining times between gels e Inconsistent labeling efficiency with radioisotopes e Inconsistent excitation emission efficiencies between different fluorescent tags e Inconsistent detection energy sources between gels during image acquisition and e Inconsistent exposure times between gels during image acquisition To accurately compare spot quantities between gels you must compensate for these nonexpression related variations in spot intensity This is called normalization With a MatchSet open select Normalize from the Analyze menu 6 23 PDQuest User Guide Matchset MSO0002 2001 10 04 X Enable Normalization Methods Basis denominator Scaling _AaO_ _ _a_a o Total quantity in analysis set PPM x 1000000 Total quantity in valid spots Percent x 100 Total density in gel image User specified Specified value Counts loaded from Gel Record Pipetting error compensation Member information
300. ion of an original scanned spot Gaussian curves are fitted to the scanned spot in the X and Y dimensions and then additional modeling is performed to create the final Gaussian spot Using Gaussian modeling you can accurately quantitate overlapping spots spots in gel streaks and multiple spots in clusters Note that spots created using the spot boundary tools section 4 6 Spot Boundary Tools are not Gaussian modeled even though they appear in the Gaussian image 4 13 PDQuest User Guide 4 4 Spot Crosshairs and Ellipses Spot crosshairs mark the centers of spots in the Gaussian image while spot ellipses show their shapes The tools for displaying spot crosshairs and ellipses are located on the Spots menu Spots Match Analysis Identification Reports Edit Spot Tools E Automated Detection and Matching ee es elo eee eee 2 288 amp ehh Se Spot Detection Wizard Spot Detection Report Add spot Remove spot More Edit Tools Cancel Restore Spot Boundary Tools Combine Show Crosshairs Uncombine Show All Grosshairs Cancelled Spots Chri F Show Elipses Combined Spots Ctr F8 Show All Ellipses Faint Spots Sine Soo Saturated Spots Alt Fy Spot Parameters F Quantity Table Shift F6 Show Quantity Al Show All Quantity Show Low Quantity Show Quality Show All Guality Show Low Quality Fig 4 8 Spots menu and Edit Spot toolbar Note that in a scanset the Gaussian image must be loaded to display crosshairs and
301. is light source if your main UV bulb fails Enter a name for your application in the Name field Click on OK to implement your changes After you have created an application you can select it from the application tree by selecting Custom and the name you created You can delete the application by selecting Custom Delete and the name of the application G 5 PDQuest User Guide Scan Dimension If an application uses trans illumination the Scan Dimension buttons become active The scan dimension is the distance traveled by the transilluminating light source as it scans horizontally across the platen Scan Dimension f 80mm 160mm 250mm 300mm Fig G 5 Scan dimension settings for trans illumination The full scanning range is 300 mm Select a smaller range if your sample is small and you do not want to wait while the light source travels over the maximum scan width High Resolution Versus High Sensitivity High Resolution and High Sensitivity are mutually exclusive options High Resolution is the normal operating mode for the Fluor S High Sensitivity provides optimal sensitivity for low light applications It 1s the default selection for the chemiluminescence application and may be selected for a custom application In High Resolution mode images are captured at the maximum resolution of the camera In High Sensitivity mode the pixels in the camera are binned 1 e four pixels are combined into one
302. is useful if you are comparing the same region of spots on multiple images and want to center the image windows on the same spot Position your cursor on a point on the image then press F3 The image will shift so that that point is at the center of the image window 3 9 PDQuest User Guide Displaying Sections of an Image To display one of nine sections of an image or MatchSet scanset go the 3x3 Sections submenu on the View gt Advanced View submenu and select a region Upper Left Middle Lower Right etc That region will be magnified in the image or MatchSet scanset Pan means to move the camera or the focus of your gel to the right or left up or down All Windows indicates that the selections you have made in the Advanced View submenu will be applied to every window in your set Location in Gel Location in Gel on the View gt Advanced View submenu displays a graphical overlay on a subwindow or all subwindows showing the region that is displayed green box relative to the entire image blue box Select the command then click the image 3 Hiding Overlays subwindow or multiple subwindows select Hide Overlays from the main toolbar or View menu and click in the subwindow or use the F4 key command in the subwindow To conceal spot markings graphs information boxes etc in a Note that by default hiding the overlays in one image of a MatchSet or scanset will hide them in all the other subwindows To turn off
303. ish background area between sliders better distinguish peaks noise from real data Frequency Distribution Fullscale Low High x Log Fig 3 16 Two views of the Frequency Distribution histogram Gamma Slider Some images may be more effectively visualized if their data are mapped to the computer screen in a nonlinear fashion Adjusting the Gamma slider handle expands or compresses the contrast range at the dark or light end of the range and this is reflected in the transform plot and preview window 3 24 Viewing and Editing Images Reset If at any time you want to return to an unmodified view of the scan data click Reset Applying the Settings Click OK to apply the selected settings to the image in the preview window Click Apply All to apply the settings to all the subwindows in the selected scanset or MatchSet or click Auto All to auto scale all the subwindows These buttons will close the Transform window and displayed the transformed image s 3 12 6 Other Transform Features Full Scale Low High The Full Scale Low High option buttons adjust how the range of image data is displayed in the frequency distribution histogram and transform plot They do not change how the image is displayed in the preview window Full Scale adjusts the frequency distribution and transform plot displays so they show the full intensity range of the image Low High magnifies the range between the low and high sliders This makes it easi
304. isplays the multiple cuts of large spots containing more than one cut Data box displays the green data area box Uncheck this box if the data area box is obstructing the view of a spot on the periphery The Compute cut overlaps function automatically calculates overlapping spot areas You can manually adjust overlapping spots by placing your cursor on the center of an overlapping spot and moving it to the left or right Compute cut overlaps Display Cut Overlaps marks overlapping spots with ellipses in the MatchSet image and the percent overlap will be displayed next to the overlapped cluster Beginning a Cut Run When all the cut spots have been aligned and confirmed in the Align Images dialog box click the Begin Resume Cutting button If you have not manually and visually confirmed the spot alignment with Jump and Confirm you are prompted to either go back and do so or proceed hi PD Quest Press NO to go back and contirm the alignment by pressing Jump and Confirm button repeated Press YES to proceed anyway This may result in reduced positioning accuracy Y Yes X No Fig 9 14 Not all spots confirmed warning If you proceed without confirmation it may result in reduced positioning accuracy You will be prompted to place a microplate in the plate holder When you click OK in the prompt box the cut run will begin The Integrated Excision tool dialog greys out while it is working 9 28 Integrated Excision Tool
305. it reaches a certain percentage of saturated pixels This percentage is set in the Options dialog box Default 0 15 percent See section B 9 Options Step IIl Acquire Image Auto Manual Live Y Expose Q Expose a couire Exposure Time sec Pe Fig B 4 Selecting Auto Expose Click on Auto Expose button appears selected Can see Exposure Time automatically changing Once an image has reached the specified percentage of saturated pixels it is captured and displayed in the video display window Auto Expose is automatically deactivated and the exposure time appears active in the Exposure Time field B 6 Appendix B ChemiDoc At this point if you are in UV or White image mode Manual Expose will be automatically activated If you are in Chemi mode the Freeze button will be automatically activated Note If you are having difficulty auto exposing your sample you can use Manual Expose to adjust your exposure time directly Most non chemi applications only require an exposure time of a few seconds which can be quickly adjusted using Manual Expose Manual Expose If you know the approximate exposure time you want you can click on the Manual Expose button In UV or White image mode Manual Expose is automatically activated after Auto Expose is complete Manual Expose active step Ill Acquire Image button appears selected O Auto y intel Expose Expose Exposure Time sec mE tie
306. iteria You can choose from three different options in the Selection Criteria area Ignore filter for spots outside current Master Filter by peak value and Filter by quality Check the Ignore filters for spots outside current master box if you are adding spots from a gel with a different PH gradient than the current Master The Master will be expanded and all unmatched spots that fall outside the area of the Master will be added regardless of quantity or quality See 5 6 Cybergels for more information on matching gels with different pH gradients The second option is Filter by peak value Use it to add all the unmatched spots or only those spots that meet a threshold peak value and or quality value To set a peak value threshold check Filter by peak value and drag the slider to the left or enter a 5 27 PDQuest User Guide threshold value directly into the box to the left of the slider As you adjust the threshold spots that no longer meet that threshold will be deselected Set the filter range by selecting the Max option button up to the maximum peak value in the gel or the Avg button up to the average peak value The third option in the Auto Add spot to Master dialog is the Filter By Quality setting This setting is similar to Filter by Peak Value except that it filters by spot quality The number of Spots selected appears below the Selection Criteria Inside master shows the number of spots that fall within the area of the cu
307. ittle effect on Gaussian noise This filter produces very little blurring if a small sized window is selected e Weighted Mean This filter is useful for reducing Gaussian noise It has little effect on salt and pepper noise e Power Mean This filter is useful for suppressing salt and Gaussian noise within an image it worsens the effect of pepper noise e ContraMean This filter is useful for suppressing pepper and Gaussian noise within an image it worsens the effect of salt To adjust the Kernel Size magnify a background region of your image using the Zoom Box tool so that you can see the individual pixels The filter size you select should be larger than the average noise feature but smaller than the smallest spots speckles If you received a message upon opening the Spot Detection Wizard indicating the detection of Ruby Speckles in your image select the Speckles tab under optional controls to access the ruby speckle filter settings Check the box labeled Apply Sypro Ruby Speckle filter to apply the filter Use the up and down arrows to adjust the sensitivity of the filter or enter a specific sensitivity in the field Click the Find Speckles button to locate the speckles in the image The speckle count field indicates the number of speckles found in the image based on the filter sensitivity To view the speckles in the image click show Speckles 4 1 d Saving Loading Parameters After you have selected the best detection para
308. ity e g black You can then fine tune the display using the High Low and Gamma sliders described below Reset Reset will return the image to its original unmodified appearance PDQuest User Guide B 6 Step V Analysis The Analysis step of the ChemiDoc acquisition window allows you to add annotations and analyze the newly acquired image Annotate Clicking on Annotate will open a separate image window displaying the captured image The default name for the image will include the date time and user if known The Text Overlay toolbar will also pop up to allow you to annotate your image The image will not be saved until you select Save or Save As from the File menu Analyze Clicking on Analyze will open a separate image window displaying the captured image The default name for the image will include the date time and user if known You can then analyze the image using the other features in the main application The image will not be saved until you select Save or Save As from the File menu B 7 Step VI Select Output The ChemiDoc window has several output options Step Vl Select Output Video Print pa Save Fig B 9 Output options Appendix B ChemiDoc Video Print Clicking on Video Print will automatically send the currently displayed frame either live or integrated to a video printer You can add information about your image to the bottom of the printout by selecting the app
309. ity of the same spot in A e Ifyou are interested in finding the spots whose quantitation has significantly changed either way either increased or decreased select the Outside limits option The proteins found in this test have increased in quantitation above the upper limit or decreased below the lower limit If you enter an upper limit of 2 0 and a lower limit of 0 33 you will find spots whose quantity in B is more than twice the quantity in A or less than one third the quantity in A e To find spots whose quantitation in B has not changed significantly from A click on the Between limits button Enter factors to define the upper and the lower limits If for example you enter an upper bound factor of 1 10 1 1 fold anda lower bound factor of 0 90 1 1 fold the analysis set will include spots whose quantity in B is between 10 up or 10 down relative to the quantity of those PDQuest User Guide spots in A Click on the Save button at the bottom of the dialog box to add the analysis set to the ASM Be sure to enter a name for the set If you forget a pop up box will remind you to do SO As with all other types of analysis sets when the Analysis Set Template is open you can view the results in the MatchSet immediately To the right of the dialog you can specify the spot marker on which gel s to show the spots and what information to show on the gel s When you open the Analysis set Template whatever analysis sets you hav
310. k Pause to pause the cut run Note that the cutter will pause only after the most recent cut has been deposited in a well Confirm the pause then click the Confirm button under the main acquisition window 9 30 Integrated Excision Tool Switching Gels When all the spots on one gel have been cut you will be prompted to place the next gel on the spot cutter platform and align that gel image with the MatchSet image see above Repeat the steps described above until the cut run is complete for each gel Ending the Cut Run Click Pause to pause the cut run Note that the cutter will pause only after the most recent cut has been deposited in a well Any remaining cut requests will remain selected in the image At this point you can click Begin Resume to complete the remainder of the cuts or abort the run When the cut run is finished the cutting tip will return to home position If you have selected the Save Validation Image checkbox the validation image will be acquired and you will be prompted to save it The validation image is a cutter camera image showing the cut spots Recutting Spots When all cuts are complete visually inspect the cut holes for visible spots or take a UV image of the MTP and confirm the cuts as follows You can then select Cut checkboxes in the cut list to recut any missed spots or cut spots that were skipped in the previous run At any time you can reopen an old cut list in the Integrated tool and perform
311. k on it again Note To highlight and magnify spots in the MatchSet without selecting the histograms shift click each histogram PDQuest User Guide PAES EBS Tool gt Hal 5tock g A P ras Save Selections D Anayiti vel Che Selections Fiber Me ri E Sena ap mo Ec by SSP COGN oO Ter C Durty A E AA O He UE 4 Fig 6 3 Reviewing spots using the Review Tool You can create an arbitrary analysis set section7 1 Analysis Sets out of spots whose histograms are selected First select the spots by clicking on the histograms If you make a mistake click a selected graph to deselect it or click the Clear Selections button to deselect all the graphs After selecting all the graphs of interest click the Create Analysis Set button and enter a name for the set in the pop up box Click Create to create the set You can then select the set in the Spot Review tool as described above To remove the set of graphs you selected for an analysis set click Clear Selections Click the Options button in the Spot Review tool to change the display options of the report When you click a spot graph that spot will be centered in the subwindow Center images on selected spot highlighted Highlight spot on images and magnified Auto zoom images by default Also by default the review tool window and MatchSet window will be displayed side by side when you open the tool Configure windows on launch D
312. ked in the Select Spot s list 7 2 4 Selecting Spot s In the Select Spot s list click on a spot SSP number to select it or SHFT CLK or CNTRL CLK click to select multiple spots that will have the same annotation entry Alternatively click the Select Spots in Image button and click on a spot in the MatchSet Master to select it To select multiple spots with the Select Spots tool ctrl click or drag on the spots in the Master Selected spots are highlighted in the list You can sort the spot list by SSP number or Name Names of spots are specified using a special category called protname which you either create or add as a standard category You type the protein name of a spot as an annotation entry in this category and you can sort by it using the Name sort option Auto select Same Entry will automatically select spots in the list that have the same entry in a specified category With the checkbox and category selected click on a spot in the list or the MatchSet the other spots with the same entry will be selected too All categories containing annotations for the selected spot will be marked in the Select Category list If multiple spots are selected no categories will be marked 7 27 PDQuest User Guide 7 2 e Entering an Annotation After you have selected an annotation category and a spot or multiple spots type the annotation in the Annotation Entry field If you are entering an annotation in a URL category it should be t
313. l Position Focus Position Q Focus Show Alignment Grid Step Ill Set Exposure Time sec E o el O Preview Q Acquire Q Live Acquire w Stop Display Options Highlight Saturated Pixels Image file size 2 60 Mb Options Help Fig G 2 Fluor S acquisition window When the Fluor S window first opens no image will be displayed The control panel has been arranged from top to bottom to guide you through the acquisition procedure There are four basic steps to acquiring an image using the Fluor S 1 Select the application 2 Position and focus the gel or other object to be imaged 3 Set the exposure time 4 Acquire the image G 3 PDQuest User Guide G2 Step Select Application To set the appropriate filter and other parameters for the type of object you are imaging click on the Select button under Select Application The available applications and their associated settings are listed in a tree that expands from left to right Mucleic Acid Gels Blotting Copper Stain Densitometry Silver Stain Custom SYPRO Orange SYPRO Red Zinc Stain Coomassie Blue Clear White Trans Fig G 3 The application tree in the Fluor S acquisition window First select your general application then select the particular stain or medium you are using If you select the chemiluminescent application under Blotting you also have the option of selecting High Resolution or High Sensi
314. l indicate whether the values are for the transmissive or reflective step tablet depending on whether you are in transmissive or reflective mode If you are in reflective mode you do not need to change the default step tablet values If you are in transmissive mode you do need to enter the correct step tablet values that were shipped with your GS 710 You can type the serial number for these values into the Tablet Serial Number field The Quantity Offset field does not apply in the GS 710 This value should be set to zero PDQuest User Guide Next enter the values for the transmissive step tablet in the appropriate fields under the Diffuse column After the step tablet is scanned the software will associate each density value with its corresponding segment on the step tablet The step tablet density values do not need to be entered each time you calibrate Once they have been entered and saved they will be automatically recalled when the calibration strip is scanned When you are finished filling out the Step Tablet Values form click on OK Diffuse Versus Specular O D In the step tablet form you enter O D as diffuse density and then the software automatically calculates the specular density Specular density is a measure of the light that passes directly through a medium Diffuse density includes light that is scattered as it passes through the medium Step tablet values are given in diffuse density but are measured by the s
315. l system e Control Gel Choose a control gel as the MatchSet Master so that it can be included in every experiment you plan to analyze and used as the Master in every MatchSet This makes it easier to compare results between different experiments e Spot Quality Editing the MatchSet Master and matching other gels to it are easier when the Master consists of high quality spots e Cybergel If you are creating a cybergel Master from gels with different pH gradients it may be easiest to add spots from each gel in sequence 1 e use the gel with the lowest pH as the template See 5 6 Cybergels 5 9 PDQuest User Guide With your Master created you are ready to begin matching and editing spots in the MatchSet see the following chapter Displaying MatchSets A MatchSet is displayed in a single window with the MatchSet members in different subwindows The MatchSet Master initially appears in the upper left subwindow and has the name Master As with scansets you can configure the subwindows in a MatchSet using the Configure Subwindows command section 3 2 Configuring Subwindows and assign images to different subwindows using the Assign command section 3 3 Assigning and Interchanging Images The Gaussian image and Filtered image if included of each member gel are both loaded in the same subwindow To toggle between the two images use Interchange Images F11 or Interchange All Images Shift F11 The member name and type of
316. lace microtiter plate on Gel Cutting Sheet Close door and click Acquire Image fh Acquire image Step 2 Examine Image Make sure that you can see a visible gel plug in each well used Step 3 Select Action Click on Specify re cuts below to repeat any cuts The Image of the microtiter plate will remain on the screen during re cut selection vf Specify re cuts gt No re cuts Fig 8 15 Image Microtiter plate window Step 2 Do not move the gel or membrane on the cutting platform 8 20 Step 3 Step 4 Step 5 Step 6 Step 7 i Basic Excision Tool Step 1 Acquire Image Light on Gel C Light off PVDF Acquire Ima EmA LET A cleric a a ric x rStep 2 Spe ANDO not move gel on stage T Addc x Multiple Place microtiter plate on Gel Cutting Sheet Max cuts st mS fh Acquire image nap to Step 3 Set x Spin whe X Spin whe No spin Backed The image of the microtiter plate will remain on the screen rStep 4 Set aa o p uv Specify re cuts X Nore cuts ation x Station Basic Excision Tool Place a clean plastic Gel Sheet on top of the gel Place the microtiter plate on top of the plastic sheet Note the orientation so that you can recognize the A1 location Close the Enclosure door Click on Acquire image when the plate is in place The UV lights will turn on and the camera will acquire an image with the same exposure ti
317. late Click the Select microtiter plate button to select a plate name size and starting well for the current cut run In the dialog box a default plate name will appear in the Plate Name field Note that the loading order of the plates are named sequentially Plate 001 Plate 002 etc by default However you can enter any name you want in the field Click the Specify Starting Well button and in the pop up box specify the first well you want to load in the current cut run If you specify a starting well other than the 9 20 Integrated Excision Tool default of Al the skipped wells will be noted in the box Click Done to make your selection Note Specifying a different starting well is essential if you want to load spots from different MatchSets or different analysis sets on the same microtiter plate You would load your spots from one MatchSet analysis set on the plate then select a different MatchSet analysis set skip the previously loaded wells The Current Run Serial field displays the serial number of the cut run The cut run serial number increments whenever you perform a previous cut run on a new MatchSet gel or gels For example if you first cut spots from one gel in a MatchSet and do a cut run then cut the same spots from a different gel in the same MatchSet and do a run the serial number for the second run would be 2 When you have made your selections click OK to complete the microplate selection 9 21 PDQuest
318. lation Mode Any of the imaging device acquisition windows can be opened in simulation mode In this mode an acquisition window will open and the controls will appear active but instead of capturing real images the window will create dummy images of manufactured data You do not need to be connected to an imaging device to open a simulated acquisition window This is useful for demonstration purposes or practice scans To enter simulation mode hold down the CTRL key and select the name of the device from the File menu The title of the acquisition window will indicate that it is simulated J 1 FX Acquisition Window To acquire images using the FX go to the File menu and select FX The acquisition window for the imager will open displaying the control panel for the imager and the scanning area window J 2 Appendix J FX i Imager FX O xi Step Select Application E ou Channel 1 Channel 2 Channel 3 Channel 4 B Xi Enable Channel C Select D rae E Radioisotopes ES i Za 1 1 CS or BI Screen Bio Rad Lowest i i F 532 1064nm EX Blank Bio Rad RI 53Znm BL s G I T T Step Il Select Scan Area H a Click and drag in diagram to set scan area j i Top 3 Left 3 1 1 1 I Bottom a Right 14 K 7 i pA 1 1 K Quadrant
319. le if you select Spots gt Spot Crosshairs with a single image displayed in a window crosshairs will simply be displayed However if you select Spots gt Spot Crosshairs with multiple subwindows displayed the command will be assigned to your mouse and you must click a subwindow to display the crosshairs in that subwindow 2 47 PDQuest User Guide 2 48 3 Viewing and Editing Images This chapter describes the tools for displaying images in windows and subwindows and magnifying and optimizing images This chapter also describes the tools for cropping flipping and rotating images and filtering image noise These tools may be found on the View Edit Image and Window menus 3 1 Windows and Subwindows PDQuest displays the multiple images in a MatchSet or a scanset in subwindows of the same image window Subwindows appear as separate smaller windows within the main image window Subwindows are designed to allow you to easily compare and edit MatchSet and scanset images at the same time 3 2 Configuring Subwindows To control how MatchSets and scansets are partitioned into subwindows click the Configure Subwindows button on the main toolbar also Ctrl F1 and Windows menu The Configure Subwindows dialog box will open The Loaded Images buttons show the types of files that are open 2D scans Gaussian images MatchSets etc and list how many images are in each type Click a button to display only images of that type in the su
320. le2 v2 x1 Raw 2 D Image Fig 6 8 Spots gt Show Quantity Replicate group quantitation can also be displayed using the Quantity Table function Shift F6 Spots menu 6 11 PDQuest User Guide Ll Quantity Table X SSF 3214 DSN i Group Average CY Count Stock 4588 6 1 E Half 151 0 4 OF Fig 6 9 Quantitation table displaying replicate group quantitation Click the spot whose quantitation you want to see If you select the All Replicate Group as your graph configuration located in the Report menu the average value of that spot s quantitation will be displayed Li Configure Graphs HalfStock E x 411 Gels All Replicate Groups Y Apply Add Edit Delete g Help Fig 6 10 Select graph configuration 6 12 Analysis Tools 6 5 Group Consensus The Group Consensus tool displays a list of all replicate groups associated with the current MatchSet The purpose of the Group Consensus tool is to use spot matching information across all of the gels within the replicate group to find possible spot detection errors Ideally spots in a replicate group would match across all gels in the group As this is not always the case the Group Consensus tool allows you to easily edit spots in the replicate group to achieve consensus across all gels When the Group Consensus Tool window opens it will be configured alongside the MatchSet window The MatchSet window displays the images belongin
321. lected only those gels with cut requests will be listed in the table Image Options Display Cuts marks each cut request with a red box in the MatchSet gel s and also shows the actual cut request circles These circles indicate the region of the spot that will be cut Note that if the Multiple Cuts Spot checkbox is selected multiple circles may appear on some or all spots Auto zoom on cut will automatically magnify each spot you select in the table Active Gels Only displays only those gels listed in the table Note that if the Show All Gels checkbox is not selected only those gels with cut requests are listed in the table and consequently the MatchSet will reconfigure to show only those gels Under Cut options the Multiple Cuts Spot option automatically calculates the size of each spot that has a cut request and if the spot is sufficiently large specifies multiple cuts of that spot Enter a maximum number of cuts per spot in the Max Cuts Spot field The multiple cut circles will be displayed in the MatchSet To customize the way multiple cuts are made for a spot click the Specify Cuts button This allows you to redraw a cut box around a large spot 9 1 c Step 3 Specify Cuts In this step you review the cut selection in the table and make changes as appropriate Note Only those gels with cut requests will be listed in the table unless the Show All Gels checkbox is selected The spots selected to be cut are highlighted in the
322. lignment Grid Step Il Image Mode UY White Chemi Step Ill Acquire Image one orz On Step YI Select Output x Highlight Saturated Pixels Invert Display E e Video Print Autoscale Reset BE El Save Fig B 2 ChemiDoc acquisition window The ChemiDoc video display window will open in live mode giving you a live video display of your sample If no image is visible make sure the camera is on check the cable connections make sure the iris on the camera is not closed and make sure that the protective cap is off the camera lens Also check to see that the transilluminator is on and working The control panel has been arranged from top to bottom to guide you through the acquisition procedure There are six basic steps to acquiring an image using the ChemiDoc 1 Position and focus the gel or other object to be imaged 2 Select Mode 3 Acquire the image B 3 PDQuest User Guide 4 Optimize the display 5 Analysis 6 Select the output B 2 Step Position Gel The ChemiDoc window will open in live mode giving you a live video display of your sample In this mode the Live Focus button will appear selected and frames will be captured and displayed at about 10 frames per second depending on the speed of your computer You can use live mode to zoom focus and adjust the aperture on the camera while positioning the sample within the area Note Newer versions of the ChemiDo
323. litatively and statistically significant using analysis sets You can compare the similarity of gels using scatter plots and review the quantitation of individual spots using the Spot Review Tool Many of these tools are interactive with the MatchSet so you can study the actual spots in the images as you review their quantities and other data You can create high level MatchSets to compare the results of different experiments And the powerful annotation tool allows you to annotate your spots link to Internet protein databases or other files and create HTML pages of spot data Spot Cutting and Mass Spec Analysis PDQuest is part of Bio Rad s ProteomeWorks protein analysis package and controls Bio Rad s ProteomeWorks Spot Cutter You can cut spots from gels or membranes digest them and perform advanced protein analysis using MicroMass s mass spectrometry instruments and software Data from MicroMass can then be imported back into PDQuest to be included in spot annotations 1 6 Publish Results Introduction When your analysis is complete you can print your experimental data or export it to another system for further analysis 1 4 Computer Requirements This software is supported on Windows 98 XP NT 4 0 and 2000 or on a Macintosh PowerPC The computer memory requirements are mainly determined by the file size of the images you will scan and analyze High resolution image files can be very large For this reason we
324. ll be disabled if you are using Optimize Exposure Click on the Optimize Exposure button A settings dialog box will open in which you can specify the total exposure time starting exposure time and number of exposures The specified number of exposures will be taken at regular intervals between the starting exposure time and the total exposure time K 12 Appendix K VersaDoc Optimize Exposure X Settings Total Exposure Time sec z0 0 Starting Exposure Time sec 1 0 Number of Exposures 5 Save Images Cancel OF Fig K 7 Optimize Exposure dialog Note You should specify no more than 10 exposures in the Optimize Exposure dialog to avoid excessive build up of image background in later exposures The fewer the exposures the less background will be added to the image See the Release Notes for additional instructions on reducing background in images captured using Optimize Exposure Select the Save Images checkbox if you want to automatically save each exposure as it is taken Click on OK in the settings dialog to begin taking exposures If you selected Save Images a Save dialog box will open in which you can specify the base file name and location of the exposure files When you click on Save the exposures will be taken The specified number of exposures will be taken at equal intervals between the starting exposure time and total exposure time The exposure status bar will show the progres
325. ll be installed when you install the main software application Make sure that your Gel Doc camera is turned on If the camera is not turned on the Gel Doc acquisition window will open and an image capture error will be displayed Simulation Mode Any of the imaging device acquisition windows can be opened in simulation mode In this mode an acquisition window will open and the controls will appear active but instead of capturing real images the window will create dummy images of manufactured data You do not need to be connected to an imaging device to open a simulated acquisition window This is useful for demonstration purposes or practice scans To enter simulation mode hold down the CTRL key and select the name of the device from the File menu The title of the acquisition window will indicate that it is simulated A 1 Gel Doc Acquisition Window To acquire images using the Gel Doc go to the File menu and select Gel Doc The acquisition window for the instrument will open displaying a control panel and a video display window A 2 Appendix A Gel Doc D Live Focus IRIS 200M FOCUS 4 Open 4 Tele Near Y Close Y wide Y Far Show Alignment Grid Step Il Image Mode UY White Step Ill Acquire Image ode ouz 1 00 E X Highlight Saturated Pixels Invert Display Auto scale Reset Fig A 2 Gel Doc acquisition window The Gel Doc video display window w
326. ll be reported as missing or excluded from calculations as appropriate This option is only available on replicate gels and classes Estimate saturated spots If a spot is saturated and therefore cannot be accurately quantified an estimate of its value is amde by fitting a gaussian to the sides of the spot and extrapolating the peak value on that basis The volume is calculated based on the extrapolated gaussian Include qualitative changes This option includes allows the inclusion of spots turned on in one gel but turned off in the other based on the minimum fold over background value you enter Note Estimate missing spots and estimate saturated spots are only available for groups and classes Choose what you wish to compare gels replicate gels or classes See Section 6 6 Sample Database for information on classes and enter your selections for comparison e g control gels versus experimental gels After you have chosen what to compare you need to decide what kind of quantitative changes you want to examine You can select Above upper limit Below lower limit Between limits or Outside limits Depending on your method choice the graph to the right changes to reflect the method The green portions of the bars correspond to the spots that will be included in your analysis set To view the scatter plot click the Scatterplot button Selecting your limits with the scatterplot is similar to the histogram in that y
327. ll mark the spots that are annotated in the selected category Click the Format button to select the style and color of the markings Show Spot Annotations will display all the annotations in the selected category next to the annotated spots in the MatchSet Click the Format button to select the style and size of the annotations 7 32 Analysis Sets and Annotations 7 3 Browsing Annotations You can click on an annotated spot in PDQuest and open an HTML page displaying all the annotation information for the spot You must have a Web browser installed on your computer to use this command a Right click on an annotated spot in the MatchSet Master and select Browse o Annotations or select the command from the Analysis gt More Annot Tools submenu or Analysis toolbar and click on a spot in the Master The default browser will open and display a page containing all the annotation information for the spot listed by category The MrpI if calculated and protein name if entered of the spot are listed at the top of the page E Human MRC 5 Fibroblasts IEF database Microsoft Internet Explorer File Edit View Favorites Tools Help Bak gt 3 A QSearch Favorites CBHistoy G5 HW S Address e C Program FilestBio Rads The Discovery Series tmp msau00031_8216 html Go Il Links Spot information SSP 8216 Annexin V lipocortin V Mr 33 3 kDa pI 4 8 Category Description Annotation Entry Li
328. lstrip if necessary If the orientation of the scanned calstrip does not correspond to the order in which the segment activities are entered in the Calstrip Record a message will be displayed Appendix L Techniques indicating that the calibration density decreases while the counts increase or vice versa To change the orientation of the calstrip so it corresponds to the Calstrip Record segment activities use the Horizontal Flip function under the Image menu If the dark segments were on the left they will now be on the right and vice versa Again we recommend that you enter your data from highest to lowest activity and display the calibration strip with the darkest segment on the left Box Segments in a Calstrip Once the calstrip is in the correct orientation go back to the Edit gt Calibrate Gels submenu and select Box Segment Go to the first leftmost segment of your calstrip Sampling boxes must be drawn beginning with the leftmost segment of the calstrip and moving right With Box Segment selected drag a box on the calstrip When you release the mouse button the area will be sampled Note When defining your box be sure to avoid sampling regions that are very close to the borders of the segment or any irregular areas 1 e areas that are overly dark or light The number of each segment will be displayed in the upper left corner of each sampling box Above the box will be a number between 0 00 and 2 00 This
329. lter Wizard 4 ie Rotate 180 tl Filter List Py AAA AA Custom Rotation Invert Data Reduce File Size Fig 3 19 Flipping and rotating commands Note These actions will erase any analysis you have performed on an image You will be asked to confirm the flip rotation before the command is executed Flipping To flip the image right to left select Horizontal Flip from the Image menu or toolbar To flip the image top to bottom select Vertical Flip 90 Rotations Select Rotate 90 Left Rotate 90 Right or Rotate 180 from the Image gt Rotate menu or Image toolbar to perform the specified rotation You will be asked to confirm your choice before the command is executed Custom Rotation If you need to rotate your image in increments other than 90 you can use the Custom Rotation command 3 30 Viewing and Editing Images Select Custom Rotation from the Image gt Rotate menu or Image toolbar A green plus sign will appear next to your cursor Click on the image you want to rotate and a circular overlay with an orange arrow will appear A small dialog box also will open indicating the angle of rotation in degrees and radians Rotate demol v2 x1 20 Angle 25 1 degrees 0 438 radians Rotate Cancel demol v2 xl 2D Scan New Fig 3 20 Custom rotation the arrow points in the direction of the new top of the image To perform the rotation position your cursor on the arrowhead and drag
330. lters Userl User2 etc cannot be renamed in the pop up list so be sure to remember which filter you insert into each position in the FX i e 690 nm filter 605 nm BPfilter To use an external laser click on the Laser button and select it from the pop up list i e 488 nm or 635 nm laser Otherwise use the default internal laser 532 1064nm Note The dual laser 532 1064 nm is not available in the FX Pro or the FX Pro Plus systems Click on the PMT Voltage button to select a standard voltage for your custom application or create a custom PMT voltage To select a custom voltage click on the Custom option In the dialog box adjust the slider to select a PMT voltage as a percentage of the maximum ll e Medium Sample Intensity selecta custom PMT voltage Low Sample Intensity Choose later Custom PMT Voltage 55 High Sample Intensity lt Lowe Sample Intensity Lowe PMT oltage High PMT oltage ot Cancel OF Fig J 6 Selecting a custom PMT voltage Note For voltages above 80 of maximum you will receive a warning message that the high voltage could damage the PMT If you select Choose Later from the list of PMT voltages the choices of sample intensity will be displayed when you select your custom application J 9 PDQuest User Guide Finally enter a name for your application in the Name field and click on OK to implement your changes After you have created an application you can
331. luminescent samples With this mode selected the data is measured in linear intensity units however the data 1s inverted so that samples will appear dark on a light background Also Chemi mode changes the behavior of the Auto and Manual Expose functions as described above B 4 Step Ill Acquire Image For many white light applications you can skip this step and save and print images directly from Live Focus mode For UV light chemiluminescent applications or faint samples the ChemiDoc control panel has several features for creating image exposures You can take an automatic PDQuest User Guide exposure based on the number of saturated pixels in the image you can enter a specific exposure time or you can take a series of exposures and select the best one Note Exposure refers to the integration of the image on the camera CCD over a period of time The effect is analogous to exposing photographic film to light over a period of time Auto Expose Use Auto Expose if you want to take a single exposure but are uncertain of the optimal exposure time Note If you know the approximate exposure time you want 3 seconds you can skip this step and go directly to Manual Expose Click on the Auto Expose button to cancel Live Focus mode and begin an automatic exposure The Auto Expose button will appear selected throughout the exposure During the auto exposure the image is continuously integrated on the camera CCD until
332. mage You can also print a copy of your merged view to a color or grayscale printer by clicking on the Print button 3 11 3D Viewer The 3D Viewer tool allows you to see a three dimensional rendering of a portion of your image This is important for such instances as determining whether a selected spot is actually two or more separate spots To see a 3D rendering of a portion of your image select 3D Viewer from the View menu Your curser turns into a crosshair Click and drag your curser over the image area you would like to view creating a box Note viewing a large area of your image may reduce performance e To reposition the box position your cursor at the center of the box The cursor appearance will change to a multidirectional arrow symbol You can then drag the box to a new position e To resize the box position your cursor on a box corner The cursor appearance will change to a bi directional arrow You can then drag that corner in or out resizing the box e To redraw the box selection position your cursor outside the box and click once The box disappears and you can then draw a new box To view the selected area position your cursor inside the box slightly off center The cursor appearance will change to an arrow Click once to open the 3D viewer 3 18 Viewing and Editing Images hi 3D Viewer Display Options Display Mode Texture Scale Factor MAME Close f Reset View 4 Help Fig 3 13 3D
333. mage Spots Match Analysis Identification Reports Window Help SR ONC RB IDEA File comma Viewing Commands Toolbars Fig 2 1 Main toolbar The pulldown menus contain e File General file commands Open Save New MatchSet Revert to Saved imaging device acquisition windows and others e Edit Text overlays Calibration Preferences Mouse commands and others e View Toolbars Zoom Box View Density Advanced View Multichannel View and others e Image Image Info Transform Advanced Crop Filter List and others e Spots Automated Detection and Matching Spot Detection Report Spot Editing Crosshairs Quantity and Quality and others e Match Automated and Classic Spot Matching Edit matching tools Cybergels and others e Analysis Analysis set functions Annotation tools MrpI Standards Replicate groups functions and others 2 1 PDQuest User Guide e Identification Excision Spot cutting and Mass Spectrometry analysis tools e Reports Graphs and Reports Print and Export e Window Tile windows Imitate Zoom Configure Subwindows and others e Help Quick Reference Guide On line Help Keyboard Layout Software Registration Below the menu bar is the main toolbar containing some of the most commonly used commands Next to the main toolbar is a status box which provides information about cursor selection and toolbar buttons 2 1 60 Main Toolbar The main toolbar in PDQuest appears b
334. mage file for example imaging device scan date image color etc For this reason the Image Report may list this information as Unknown for imported TIFF files Video Printing The Video Print command allows you to print images and reports on a video printer To create a video printout of the active window select Video Print from the File gt Print submenu Note Video printing requires installation of the video board and cable that come with the Gel Doc 1000 2000 and ChemiDoc gel documentation systems The video board and cable can also be ordered separately 2 24 General Operation Settings for the Mitsubishi P9OW P91W Video Printer There are three settings for the Mitsubishi P9IOW P91W video printer Set Contrast to O Brightness to 0 and Gamma to 5 The dip switches should stay in the orientation in which they are shipped Pin 1 is up on and Pins 2 10 are down off 2 6 Exporting You can export gel images to TIFF images and export MatchSets and Annotations using the Export submenu in the Reports menu File Edit View Image Spots Open Chri4 o Save Chris Save AS Save All Ctrl Revert to Saved Close Alt F4 Close All Unload Ctrl I Gel Doc Chemi Doc 65 300 Personal Fix Ea Print Export Export to TIFF Image Exit Ctrl g Export Matchset Basic Export Matchset Export Annotations Fig 2 16 Export submenu 2 25 PDQuest User Guide 2 6 a
335. mage gt Image Info displays general information about your image including the scan date scanner used scan area number of pixels in the image data range and the size of the file Gaussian image info includes the number of spots total data in the spots and other information There is also a field where you can type in a file description or comments i stocki v1 x1 Raw 2 D Image AN E 3 x Bee Program Files Bio Rad File The Discovery Series Info Sample Matchsets _Half5tock Data stock vl x1 gsc Description 10 54 dave Scan date unknown Scanner GS 710 Scan area mm 183 3 Y 143 9 Scan plselz em 1015 f 7a Fisel size um of 180 5 Y 180 6 Data range 0 0 2 00 0D Memory size 791 93 Eb 9 Jul 1999 08 56 Horizontal Flip ipo S eh le S gt Horizontal Flip X Cancel ll Help Fig 2 11 Image Info box History The Image Info dialog displays differently depending on the type of file you are acquiring information about for example Raw 2 D or Gaussian History lists the PDQuest User Guide changes made to the image including the date the changes were made To print the file information click Print 2 3 0 Reduce File Size Scan files can be quite large and computer systems do not have unlimited memory or storage space If you are having difficulty loading or storing a particular scan you may want to reduce the size of the file by reducing the number of pixels in the imag
336. mated Detection and Matching Detect Spots dialog box You can also open the dialog directly by selecting Automated Detection and Matching tool form the Spots menu All currently loaded gel scans will be listed in the panel 4 9 PDQuest User Guide 1 Automated Detection and Matching on disk ay A x Step 1 Select Gels To Analyze Step 3 Create MatchSet Add samples Name Auto sample4 Description ES Remove sample2 Comments sample3 RS Master sample Directory c Program Files Bio Rad The Discovery Series Sampl Change Member sharing Do not make copies refer to the originals Copy the originals into this matchset s data folder _ Disable this step Count Es i Dre items to reorder them Step 4 Match Gels Parameters Default Match Settings 00 Edit Step 2 Detect Spots on Gels Disable this step Parameters AA Edit Log File Name KTro11_20011010_1122 3 Go X Cancel Fig 4 5 Automated Detection and Matching Automated Detection dialog The Automated Detection and Matching tool combines several processing steps into a single operation This is a new feature in PDQuest and should become a part of your normal workflow Step 1 Select Gels To Analyze Use the Add button to select gels to be analyzed and matched Unlike previous versions of PDQuest you do not have to open the images before processing them The Add butt
337. mber of additions and changes that have been made x A O annotations were added or changed E Fig 7 15 Changes made indicator Click OK 7 2 g Reviewing Annotations in the MatchSet The Image Options in the Annotation Tool allow you to highlighted selected spots in the MatchSet Master You can also display actual annotations on the Master image Note When reviewing annotations in the MatchSet it is useful to shrink the annotation tool window Click the arrow button in the lower left hand corner of the Annotation Tool to collapse the tool only the reviewing functions will be displayed 7 31 PDQuest User Guide fh Annotation Tool meau00031 Ea Select Category anne xine ir autoantigen calhinprot i cellcycle cellsiqnetdatah cellulloc m cellulrole chaperonin Add Edit Delete Std Show description Image Options x Highlight target spot Mark all annotated spots o Format x Mark spots in category Format Format Bote lt Click here to expand tool Fig 7 16 Annotation Tool in reviewing mode Show spot annotations Highlight Target Spot will highlight the spot s that are selected in the Annotation Tool The spots will be marked with Mark All Annotated Spots will mark all annotated spots in the MatchSet Click the Format button to select the style and color of the markings Mark Spots in Category wi
338. me as the first spot cutter image Assess the image on the screen for the wells that need to be recut Step 1 Setup MTP Cover gel with Gel Cutting Sheet Close door and click Acquire Image Make sure that you can see a visible gel plug Step 2 Examine Image ineach well used Step 3 Select Action Click on Specify re cuts below to repeat any cuts during re cut selection Ge Fig 8 16 Microtiter plate with fluorescent spots in wells Use the Zoom and transform functions on the right side of the Image Microtiter Plate window to get a better view of the microtiter plate wells 8 21 PDQuest User Guide lil Basic Excision Tool Step 1 Acquire Image Light on Gel iaht off PVDF Acquire Ima MALETA RdA AO Step 1 Setup MTP ANDO not move gel on stagel Cover gel with Gel Cutting Sheet Place microtiter plate on Gel Cutting Sheet RDA 6 9 0 POONA 001070 SOS BxJMukiple1 close door and click Acquire Image Max cuts sp nS 9 9 Ie x Snap to IO Step 2 Examine Image OK gt Step3 Set Make ree i i can see a visible gel plug OS in each well used X Spin whe o AI x Spin whe eae Oo Step 3 Select Action No spin Click on Specify re cuts below to repeat any cuts SOS oo or Backed The image of the microtiter plate will remain on the screen BS oo PEA during re cut selection 81
339. me spot in that member it will appear marked with a box Click it to place the landmark Landmarked spots are marked with green triangles 9 15 PDQuest User Guide Hatch Tools Ape GB BMA T R hi Matchset demo Modified Landmarks Count 1 Landmarks Count 1 Landmarks Count 1 STD derno std dernol Modified demol vl ci demod vl Filtered A Landmarks Count 1 Landmarks Count 1 demos vl Filtered demod vl Filtered Fig 5 11 Landmarking a spot If the program is unsure about positioning the landmark the area in which the spot was expected will be circled Locate the appropriate spot and click on it As each landmark is placed the cursor will jump to the next gel Landmark the spot in all members of the MatchSet except when creating a Master out of gels with different pH gradients see 5 6 Cybergels If you accidentally landmark the wrong spot in a gel simply go back and click the correct spot The landmark will move To remove a landmark from all the MatchSet gels select Unlandmark from the Match menu or toolbar and click the incorrect landmark in the Master To remove a landmark from a single gel click in that gel To highlight all the landmarks in a MatchSet use the Mark Landmarks command F8 Match menu Match toolbar in the Master image As you add landmarks study the auto matching in all regions of the gels Add more landmarks to regions where auto matching has missed spots
340. message Saving the Image After the scan is complete a message will appear asking you if you want to keep the scan If you select Yes a separate window will pop up containing the new image You can then save and analyze the image using the standard menu and toolbar functions Appendix I Personal FX 1 5 Options Auto Save After Scan To automatically save any scan you create click on the Auto Save After Scan checkbox Note In PDQuest this option is preselected and cannot be turned off All images must be automatically saved when acquired With this checkbox selected when you click on Acquire a Save As dialog box will open asking you to specify a file name and location for the image you are about to create The scan will begin when you click on the Save button Make Backup Copy You can automatically create a backup copy of any scan you create To do so first select Auto Save After Scan see above then select the Make Backup Copy checkbox With this checkbox selected when you save a scan a backup copy will be placed in the same directory as the scanned image Windows backup files will have an sbk extension Macintosh backup files will have the word backup after the file name Note To open a Windows backup file you must first change the file extension To locate the file after changing the extension select All files in the Files of type drop down list of the Open dialog Highlight Saturated Pixels Wh
341. meters for the image save them by clicking the Save button next to Parameter Set below the image window Enter an appropriate name for the set e g the type of stain used in the gel in the pop up box Detecting and Editing Spots Click OK The parameter set will now be available for processing all similarly detected gel scans To load previously saved parameters click the Load button and select from the list of parameter sets When the parameters are loaded you can proceed to Find Spot Centers on the loaded image Each parameter set is saved as an adp file in the Discovery Series lt user name gt prm directory To delete a parameter set delete its adp file 4 1 e Processing Gels Click the Process All Gels button to close the Spot Detection Wizard and open the Detect Spots dialog box If you have not saved the current detection parameters you will be prompted to do so before you exit the Wizard The Quit button will close the Wizard without opening the Auto Detection and Matching window If you have not found spot centers any unsaved parameters will be lost 4 2 Detecting Spots After you have created and saved a set of detection parameters using the Spot Detection Wizard you are ready to detect spots on your images In general you can use the same parameters to detect spots on all gels that use the same stain and have been run and scanned in the same manner Click Process All Gels in the Wizard to open the Auto
342. mporary license Your license will expire on The temporary license is designed to give you time to purchase the software if you have not already done so Software License 2 Welcome to POQUEST Current License Check License our license will expire on 05 Apr 2001 Aun Enter Password Check License On line IF your registration has been updated with Bio Rad this Registration Form Will update your password and service will continue Exit POQUEST Single system license License expires on 05 Apr 2001 without interruption Aun Aun the program as usual Help Fig 1 9 Temporary license screen During the 30 day period the Software License screen will appear every time you open the software To use the software during this period click on the Run button Network license holders can click on the Check License button at any time during the 30 day period to activate their full network license If your network license is not activated when you click on Check License notify your network administrator HSK users have 30 days to purchase the software and obtain a purchase order number and software serial number from Bio Rad When you have this information click on the Check License or Registration Form button in the Software License screen to register your software 1 13 PDQuest User Guide Software License Registration Form 3 xi Please fill out the form below to authorize full use of your s
343. n you change one imaging area dimension the other will change to maintain the aspect ratio of the camera lens The imaging area will change depending on your zoom factor For example if you have zoomed in on a area that is 4 5 x 3 5 cm then you would enter 4 5 for the width 3 5 for the height would be calculated automatically Note Your imaging area settings must be correct if you want to do 1 1 printing They must also be correct if you want to compare the quantities of objects e g using the Volume Tools in different images The imaging area dimensions also determine the size of the pixels in your image 1 e resolution A smaller imaging area will result in a higher resolution G 6 e Auto scale Transform Auto scale Transform after Acquisition allows the user the option of having the image automatically perform the Auto Scale transform function upon completion of image acquisition This eliminates the need to transform an image or re scan an image when the acquisition time was too short or the iris not opened enough To enable the auto scale transform function check the box labeled Auto Scale Transform after Acquisition in the Options dialog G 7 Other Features Display Options x Highlight Saturated Pixels Image file size 2 60 Mb Options Help Fig G 11 Other Fluor S acquisition window features Appendix G Fluor S Highlight Saturated Pixels When this box is checked any saturated pixels in the image will
344. nalysis Set Arhit Demo 76 spots HS NT Ll Excision Gel Selection H5 L Step 1 Select Analysis Set Set name Quant Demo gt Step 2 Set Selection Mode f Auto select Tolerance ratio 1 50 a hd Gels selected e Qual Demo 106 spots Quant Demo 23 spots Cancel Single gel Include spot qualit Single group Ej Sn f Manual Image options Multiple cut options x Display cuts Table options O Show quality Show ratio x Show all gels x Enable multiple cuts spat Max cuts spot E Specify cuts X Suto 00m on cut Active gels only Step 3 Specify cuts SSP Demo 3Odegreel Demo a Demo 30degree Demo 0 01 955546 3 2600792 0 1304 489535 1 FE n mi En En cm Cc En 2505 2301 us Totals O Proceed yf Close x Cancel H Help 2955080 0 1180016 9 376953 3 1482125 8 152945 2 1692065 9 311594 7 32045395 0 1467975 4 414356 7 2247 239 0 2076525 3 1204492 6 370293 7 2451871 3 1456325 6 El Me E ar 45 af 30 El Fig 9 2 Excision Gel Selection 9 1 a Select the analysis set you want to cut from the pop up box The Excision Gel Selection dialog will display a table of the spots in the analysis set and their quantities step 1 Select Analysis Set 9 3 PDQuest User Guide in the different gels in the MatchSet The first spot in the set is magni
345. nce and create a new one under current conditions None If you do not want to perform dark subtraction select None No dark exposure will be acquired or subtracted H 6 b Live Exposure Mode Exposure Count If you are using the Live Acquire function see previous section you need to specify how many intermediate exposures you want to view save during acquisition Enter this number in the Exposure Count field The total exposure time will be divided by the number you enter in the Exposure Count field If you enter an exposure time of 10 minutes and a count of 10 you will create 10 intermediate exposures at 1 minute intervals Note Do not enter a count that will result in an intermediate exposure time that is less than the minimum exposure time for the mode you are in The minimum exposure time in trans illuminated mode is second and the minimum exposure time in epi illuminated mode is 0 1 second Example For a trans illuminated application an exposure time of 20 seconds and an exposure count of 21 would result in an error Save All Intermediate Images If Auto Save After Scan is selected see following section the Save All Intermediate Images checkbox will become active If you select this checkbox all your intermediate exposures will be saved as separate files These files will have the same Appendix H Fluor S MAX root name appended by a number indicating the exposure sequence The final full exposure will have th
346. nce you have created a gel sample association you can add attributes to the sample Highlight the sample and click Add attribute This opens the list of available 6 19 PDQuest User Guide attributes Select the attribute or attributes you wish to add to the list and click Done Or if you wish to create a new attribute click New i Select Sample Attribute X temp spotnumber x Apply when possible wf Done 1 Mew X Cancel 1 Help Fig 6 16 Adding an Attribute Once the attribute has been added to the table you can add a value by clicking the cell If you enter a new value for the attribute a warning dialog appears asking if you would like to add the value to the attribute Check the Apply when possible box to automatically add the attribute to any samples that do not already have the attibute added This feature allows yo to quickly apply an attribute to a great number of samples All you need to do from there is enter the values 6 6 b Classes Attributes are variables that cannot be measured but must be expressed in a qualitative manner A class is a group of gels with the same attribute value An example of this might be a MatchSet of six gels Some of these gels may be treated or untreated By attaching these values to your sample in the sample database you can refer to these sample as a group of treated or untreated samples You can also create 6 20 Analy
347. ncel Fig N 12 Select Microtitre plate Select plate to edit A given cut run may fill several plates Select the plate of interest here Plate name Select or create the Microtiter Plate record by pressing the Plate name link Plate size This 1s determined by the plate you selected above First free well This is determined by the selected plate If you wishh to change the starting well click the Specify starting well button Click the OK button to proceed with the excision step which when completed fills the wells with gel plugs Note You may not re use wells of an existing plate however you may use empty wells on an existing plate WorksBase requires that each plate have a unique identifier such as a barcode and that each well be used no more than once N 7 b Digestion The digestion step must be recorded in WorksBase for Proteomics If you open the Program Project Experiment Excision step you will see the container for the microtiter plate If you right click on the container you will see the command to digest the samples spots Perform this step It will result in a MALDI plate another type of container The next step is to set up the plate for mass spectrometry MS or MS MS N 19 PDQuest User Guide N 7 c Identification To successfully identify proteins you must have ProteinLynx Global server version 1 1 or Matrix Science s Mascot Create a FAST A file using WorksBase for Proteomics The PLG
348. nction Display Segment Quan Above each sampling box will be displayed the average O D value within the box and just below the box will be displayed the corresponding DPM or other specified unit This should tell you which sampling boxes correspond to errant data points on the graph Usually problems arise when a box straddles the boundary between two adjacent segments or the density is very uneven within the segment due to bleeding from an adjacent segment These sorts of problems should be remedied by redoing the sampling boxes Use the function Remove Segment to remove an errant sampling box Draw a new sampling box on the segment using Box Segment as described above Place the new box carefully to avoid repeating the problem For example keep the box away from segment boundaries If the segment shows bleeding place the smallest box possible in an area of the segment with representative O D Try not to sample blotchy areas Replot the calibration curve using the function Plot Calib Curve If there is still a problem you can opt not to include that particular segment in the calculation of the calibration curve Select De Activate Segment and click on the problem segment The segment will still exist so its counts in the corresponding Calstrip Record do not need to be changed or deleted However the segment will not be included in the calculation of the calibration curve If you change your mind and want to reinclude the segmen
349. ndow for the densitometer will open displaying a control panel and a scanning window F 2 Appendix F GS 800 Densitometer GS 800 Calibrated Transparency Step Select Application 1 Select 3 X ray film Gray film 7 Fiter Red X Green JBlue White 9 Light Reflective Transmissive 41 13 Step Il Select Scan Area Al Preview scan z 17 Click and drag in diagram to set scan area Top o o Left o o 19 Bottom lo o Right lo O Fs Step Ill Select Resolution X resolution 63 5 Microns Select 27 Y resolution 63 5 Microns z 29 Image file size 0 00 Mb E 33 Obie Y suo s Options 37 J uto Save After Scan Hide Grid 39 1 23 25 27 2 Highlight Saturated Pixels O Options a Help Fig F 2 GS 800 acquisition window The scanning window is marked by grid lines that divide the area into square centimeters These are numbered 1 40 top to bottom and 1 30 left to right if the light source 1s reflective and 1 40 top to bottom and 1 29 left to right if the light source is transmissive To hide the gridlines select the Hide Grid checkbox under Options Below the main scanning window is the calibration strip window Every
350. nel sequential scanning This allows you to automatically scan the same object using up to four different applications e g to detect different types of stains on the same gel First you select a channel then you select the application under that channel J 2 a Selecting a Channel The four channels are accessed using the tabs under Step 1 Channel 1 is always enabled that is the FX will always scan using the application settings selected under Channel first To enable any of the remaining channels click on a channel tab then select the Enable Channel checkbox and select the application for that channel as described in the following section Appendix J FX Step Select Application Channel 2 Channel 3 Channel 4 Click on the tab to select the channel Select the checkbox to enable the channel Click on the Select button to select the application Densitometry Coomassie Blue Gel Blot Medium S532 1064nm EX Blank 555nm LF 1l06 4nm BL Fig J 3 Enabling Channel 2 Enabled channels have a green check mark on their tabs You do not need to enable Channels 2 4 in sequence For example you can set up your four most common applications using the different channels but only enable Channel 4 for a particular gel Channel 1 would be scanned first Channel 4 second Enabled channels are scanned sequentially The separate scans are displayed and saved as separate images The total scan
351. ng and Editing Images image detail The crosshair will make it easier to position the box in the other images so that it encloses the same area 3 Select Save Crop Settings from the submenu enter a name for the current crop settings in the pop up box and click Apply 4 Complete the crop action in the current image by positioning your cursor inside the box slightly off center and clicking to perform the crop as described in Cropping Images 5 Open or select the next image you want to crop select Load Crop Settings from the submenu and select the name of the settings you saved The crop box and crosshair will appear on the image 6 Reposition the crop box so that the crosshair is correctly aligned with the appropriate image object then complete the crop as described in step 4 above To delete any crop settings you have saved select Delete Crop Settings from the submenu and choose the settings to be deleted from the list 3 14 Flipping and Rotating Images The matching functions in PDQuest will tolerate minor distortions in the shape and orientation of images However if your images are grossly misaligned you may have to flip and or rotate them These commands are located on the Image menu and toolbar 3 29 PDQuest User Guide nage Spots Match Analysis Image Tools x Image Info Ctrl I El Transform Ctrl B Crop Advanced Crop d Vertical Flip Horizontal Flip Rotate Rotate 90 Left i l Rotate 90 Right Fi
352. ngel 226229 Exposure start 25 Jul 1989 21 00 lt Duration days 1 558 0 Fig L 3 Gel Record Editor Enter the date on which the gel was run in the text box next to the Gel run date prompt Next to the Calstrip prompt click on the button that corresponds to the kind of calibration associated with this gel In most cases you or someone in your lab has made the calstrips so the button labeled User Batch is the appropriate choice Next to the User Batch prompt click on the arrow From the list displayed select the calibration record name for the calstrip The Detection and Isotope fields are automatically obtained from the specified Calstrip Record Click on the appropriate button to indicate the type of detection used for your gel 1f not already defined from the batch file Click on the Isotope button to indicate the isotope used in the gel This 1s usually though not necessarily the same isotope as the one used in the calstrip Next enter the number of counts loaded on the gel Enter the day on which you began exposing the gel to film in the text box next to the Exposure start prompt L 5 PDQuest User Guide Note Most calibration problems arise from incorrectly entering the dates used to calculate radioactive decay factors for the isotopes Wrong values can make the calstrip appear to have decayed to nothing Next to Duration days enter the number of days that the film was exposed Once this data has be
353. ning time depends on the number and type of enabled applications If you have selected Auto Save After Scan the image created using Channel 1 will have the base file name and images created using subsequent channels will have the base file name plus a version number v 2 v 3 v 4 Note that the image version number does not necessarily correspond to the channel number For example if you scanned an image using only Channels 1 and 4 the image created using Channel 4 will be saved as version 2 v 2 J 2 b Selecting an Application To select an application 1 e the appropriate filter and other parameters for the type of object you are imaging click on the Select button under a channel tab J 5 PDQuest User Guide Radioisotopes Chemiluminescence Chemifluorescence ONA Stain Gel Protein Stain Gel Svpro Orange Svpro Red Microtiter Plate l High Sample Intensity Mile Red l l Densitometry Medium Sample Intensity Sypro Ruby l Custom Low Sample Intensity Fluorophores Fig J 4 Example of an application tree Ethidium Bromide gel Standard Applications The standard applications and associated settings are listed in a tree that expands from left to right When you select a standard application the software automatically selects the appropriate filter s in the FX for that particular application Some applications are inactive in the FX Pro and FX Pro Plus systems Standard FX Applications CS or Bl Screen B
354. nk protname Protein Name asdfasdfasdf swissprot Swissprot annexins Annexin autoantigen calbinprot Calctum binding protein cellcycle cellsignetdatab cellulloc Cytoplasm cellulrole Cellular Role According Transport to EGAD chaperonin comigration Comigration with Mouse Proteins comments cytoskelprot Cytoskeletal protein y E Done g My Computer Fig 7 17 Example of the annotation browser 7 33 PDQuest User Guide If you have created an annotation link for the spot in a URL category see section 7 2 6 Annotation Categories that link will appear in the page under the Link column Click on this link to open the database or Web site specified by the category and link to the protein information Use the Print command in the browser window to print the annotation information The displayed page is a temporary file that can be saved by using the browser s Save As command You can then include saved pages on your Web site or e mail them to colleagues 7 4 Creating Annotations from Analysis Sets Analysis sets are used to group spots in a MatchSet that are statistically and scientifically meaningful After you have created an analysis set you can create an annotation describing the spots in the set Note You can create annotations for individual spots or other groups of spots using the Annotation Tool With the MatchSet open select Create Annotation from Set from the Analysis gt More Ann
355. nly require an exposure time of a few seconds which can be quickly adjusted using Manual Expose Manual Expose If you know the approximate exposure time you want you can click on the Manual Expose button Manual Expose is automatically activated after Auto Expose has deactivated A 6 Appendix A Gel Doc Step Ill Acquire Image Manual Expose active button appears selected Auto Manual Expose Expose Exposure Time field active adjust number of seconds Exposure Time sec z 00 l l oO Freeze Fig A 5 Manual Expose With Manual Expose activated you can adjust the exposure time directly by changing the number of seconds in the Exposure Time field Type in a number or use the arrow buttons next to the field When the specified exposure time is reached the last captured image will be displayed in the Gel Doc image window The camera continues to integrate the image on the CCD updating the display whenever the specified number of seconds is reached Once you are satisfied with the quality of the displayed image click on the Freeze button to stop the exposure process The last full exposure will be displayed in the image window Step IIl Acquire Image Auto Manual P Expose Q Expose Exposure Time sec E Freeze Fig A 6 Freezing the exposure Click on Freeze to stop the exposure process A 7 PDQuest User Guide Note Freeze is automatically activated
356. no nono no nonnnnnnnrnnnrnnnrnnrnnn F 6 POTS sted eas asa e OS F 14 DA e o E A F 14 Preview E asi vive cdie sasuaddAcanecesaued F 7 Resolution SEE CUS rca F 8 Saturated pixels highlighting oooccccccncnnnnnnnncnnnnnnnnnnnnnnnnnnnnnonononnnnonononos F 15 AM A OIO E F 7 Si O o S F 13 SCT SW Id Wide iSewletas F 3 O acne F 1 SUMMA ON MOdE salino iia Oro F 2 Hardware Protection Key INFACTINUOSING aria aii 1 11 PO A douse vadneasuiaa dues 1 10 AA e N dedohu 3 10 Pish level Matches ica talas 5 34 A R eee abe casa 5 35 VAM SECTAS ANO LAOS A aos 1 37 Transkein mne LD SINS adas 6 36 Transterrine MP dada tad cds 6 31 SPT AMISTCTIING SS PS A A adavecstasveahedenicvcascanelac 6 34 FATS CO STA a Maat adie ton 11 4 Mas TIMO AO Ai 2 17 TASS repor PINUS ne E EAN 2 23 Index 9 PDQuest User Guide Mina Se Slack Tool aii cis 6 5 Image SLACK OOM a O OS 6 4 Images ASe nine TO MIOS i 3 3 A A A O ta Sametime eave Tenet meee ets tee 2 15 EX POLIO do 2 26 Ere lal AN ON aires drenado 3 3 O 2 10 SAM a lib ie DEN 2 16 O 6 5 TOPES aii lll coloco E E aora 2 7 aite LO lille licda 3 7 Import Mass Spec results from WorksBase cooccccccccccncnnnnnnnnnnnonononononononananannnos N 21 imporune TIFE MeS aio aba raya treater 2 12 Installation dd AG Lo YO Los y CPAP Ro PT 1 9 WINGO WS ONE E A STN 1 9 Ea ETEA Manne E cate A E E E A N A E E T 2 36 Integrated EXCISION Too b aeranta EN OEE N secs 9 9 AHMAS IMASE dra 9 22 BESITOS a a ee
357. ns Plot Mrpl Grid Plot SSP Grid Ctrl F9 Transfer Annotations Display 55Ps AIL ES Remove Mrpl Data Display All SSPs Transfer Mrpls Transfer 55P5 Assign DSN Grid Plot OSM Grid Plot DSN Grid Counts Display DSNs Display All DSNs Transfer DISMs Fig 6 1 Analyze tool and submenus 6 1 Spot Review Tool The Spot Review Tool displays histograms of all the spots in a MatchSet The bars in each histogram represent the spot s quantity in each member of the MatchSet If 6 1 PDQuest User Guide Replicate Group quantitation is selected the bars represent the spot s quantity in each group Seel1 2 Histogram Graphs for a more complete description of histograms This tool is interactive so you can use the graphs to identify spots in the MatchSet for matching and editing or create an analysis set out of selected spots With a MatchSet open select Spot Review Tool from the Analyze menu or View gt Toolbars gt Graph Tools toolbar The review tool will open and the MatchSet will be reconfigured alongside it This automatic window configuration can be set under the report options see below i Spot Review Tool mpdi00187 f All spots Analysis set Filter No filtering Input Sortby 6 S55P DS5N e Trend 7 Quantity Page number 104 of 108 tet First Prev pr Next ai Last ar Un narm DPM SSP 8316 38 Un narm DPM I I SSF 5406 Ba Un narm DPM SSF F218 qa n normi DPM
358. nsensus Summary table highlighted in blue The global Actions affects all the spots in the selected cell while the Individual Review tool allows you to review each spot individually and add or remove the spot represented by a blue triangle in the gel images 6 5 a Review Individual Spots The Review Individual Spots section of the Group Consensus allows you to individually review each spot in the group The active group field shows the spot number of the highlighted cell in the Consensus Summary table Use the arrows to 6 15 PDQuest User Guide cycle through the spots in the active group The Review Tool displays a quantity graph of the selected spot The bars in the graph represent the spot s quantity in each member of the group where it appears To select a different group to review select a different cell in the Consensus Summary table To select a spot in a gel click the button labeled Select Spot in Gel then click on a spot in one of the gel images The spot is automatically selected and the group to which it belongs is highlighted in the Consensus Summary table To see spot number in the gel check the box labeled Display SSPs When this box is checked the spot number appears below the marker in the gel 6 5 6 Achieving Consensus The following actions are used to make changes to the replicate group to achieve consensus across all members in a group Automatic Auto Add Allows you to automatically add th
359. nterface SCSI cable To use the GS 700 you must have a SCSI card installed in your PC If you have a PC with a Windows 98 or Windows ME operating system you may also need to load the SCSI and WinASPI drivers that came with the card Simulation Mode Any of the imaging device acquisition windows can be opened in simulation mode In this mode an acquisition window will open and the controls will appear active but PDQuest User Guide instead of capturing real images the window will create dummy images of manufactured data You do not need to be connected to an imaging device to open a simulated acquisition window This is useful for demonstration purposes or practice scans To enter simulation mode hold down the CTRL key and select the name of the device from the File menu The title of the acquisition window will indicate that it is simulated Note There is no simulated calibration for the GS 700 and GS 710 Imaging Densitometers D 1 GS 700 Acquisition Window To acquire images using the GS 700 go to the File menu and select GS 700 The acquisition window for the densitometer will open displaying a control panel and a scanning window Appendix D GS 700 lt Densitometer GS 700 Uncalibrated Transparency Fig D 2 GS 700 acquisition window The scanning window is marked by grid lines that divide the area into square centimeters These are numbered 1 33 top to bottom and 1 21 left to right if
360. ntitation of a cluster of spots into one spot This can be performed on Filtered images in a MatchSet or scanset Select Combine from the Spots gt More Edit Tools submenu or Edit Spots toolbar and then draw a box around the cluster of spots that you want to combine in the Filtered image After you draw the box click on the point in the box where you want the combined spot to be placed The cluster will appear as one spot in the Gaussian image it will still appear as individual spots in the Filtered image but the quantitation will be combined To resolve a combined spot into its original cluster select Un combine from the submenu or toolbar and draw a box around the combined spot in the Filtered or Gaussian image The spot will be uncombined into its original multiple spots Combined Spots Ctrl F8 Spots gt More Edit Tools submenu Edit Spots toolbar highlights all combined spots in the MatchSet 4 9 Saturated and Faint Spots Saturated Spots Spot detection may not correctly fit spots in saturated regions of the image Therefore although saturated spots will appear in the Gaussian image they are not quantified using Gaussian modeling their quantity will be reported as zero You should use the spot boundary tools section 4 6 Spot Boundary Tools to quantify these spots To highlight spots that are saturated in the image use the Saturated Spots command Alt F7 Spots gt More Edit Tools submenu Edit Spots toolbar in any image
361. o copy the DSNs from one higher level MatchSet member to the others This makes it easier to correlate data from the member MatchSets With the higher level MatchSet open select Transfer DSNs from the submenu In the first pop up box select the MatchSet member you want to copy from in the second box select the member you want to copy to You will be asked to confirm the transfer When the transfer is complete the number of DSNs transferred will be listed in an alert box Repeat the procedure to apply the numbers to all the members 6 36 7 Analysis Sets and gt Annotations Analysis sets and annotations are tools to help you organize and analyze your spot data Analysis sets allow you to create groups of spots that are statistically and biologically significant You can also create sets of spots to cut using Bio Rad s ProteomeWorks Spot Cutter Annotations allow you to add notes comments and other pertinent information to spots Using annotations you can create links to protein databases or files import mass spec data and store spot information in HTML format for easy portability and uploading to Web sites 1 Analysis Sets The analysis set tools allow you to create and study sets of proteins that are statistically and scientifically significant You also use analysis sets to identify spots to cut using Bio Rad s ProteomeWorks Spot Cutter There are six different kinds of analysis sets Qualitative Analysis Sets A
362. o deselect it Selecting the Auto Scale Image When Assigned checkbox will automatically adjust the brightness and contrast of each loaded image based on the data range in the image It invokes the Auto Scale command from the Transform window when an image is first opened in the viewer Note that this setting affects only how the image is displayed in the viewer not the actual data Note If you deselect this checkbox any images currently displayed will remain auto scaled click the Transform button in the viewer and click the Reset button in the Transform window to undo auto scaling Buttons for various viewing tools are included in the Multi channel viewer Commands such as Zoom Box and Grab will change the display of all the images in the viewer at once Note that if you open the Transform window you can adjust the display of each channel independently by selecting the appropriate channel option button in the Transform window Similarly the Plot Cross Section command will report the intensity of each channel separately 3 17 PDQuest User Guide Exporting and Printing You can export a 24 bit TIFF image of your merged view by clicking on the Export button This will open a version of the Export to TIFF dialog Note that you cannot export image data from the Multi channel Viewer only the current view of the image designated as Publishing Mode in the Export dialog The colors in the viewer will be preserved in the exported TIFF i
363. o scan click on the Acquire button The scanned image will begin to appear in the scanning window line by line To interrupt a scan click on the Stop button A message will ask you to confirm the interrupt and then you will be asked if you want to keep the partial scan This feature is useful if you overestimated the size of the area you selected After the scan is complete a window will open displaying the scan image at which point you can analyze and save it Note The image will open with a default file name that includes the date time and if applicable user name However unless you have selected Auto Save After Scan the file will not be saved until you select Save or Save As from the File menu E 13 PDQuest User Guide E 7 Other Options Oversample This feature allows you to scan at the maximum resolution of the GS 710 42 3 x 42 3 microns and then use spatial averaging to create an image with lower resolution This can result in better images at lower resolution however it takes longer to scan To turn on oversampling click on the More Options button in the acquisition window and click on the Oversample checkbox With oversampling on you can specify your own resolution within the range of 43 169 microns by entering values directly in the fields next to X resolution and Y resolution in the main acquisition window Auto Save After Scan To automatically save any scan you create click on the Auto Save After S
364. o the well The tip should be centered inside the well Click Park to return the tip to its home position Wash Station 1 position Calibrate the position of Wash Station 1 on the cutting platform by setting the cursor in one of the Wash station fields and pressing enter Then follow the steps described above for Well Al Once you have set the position of station 1 the cutter robot will automatically calculate the positions of the remaining wash stations 9 14 Integrated Excision Tool Miscellaneous Settings Specify the diameter of the cutting tip by selecting 1 0 mm or 1 5 mm Note that the cut request circles that you make on the image reflect the approximate size of the cutting tip and will change based on the tip size selection If you are using the ProteomeWorks spot cutter you will need to crop the left margin of your image Crop Left Margin repositions the image in the Excision Tool window to the left Enter a value in screen pixels in the field to reposition the image Note that you must click Done to close the settings dialog and then acquire a new image for this change to take effect Note that cropping the left margin does not change the raw image only the portion of the displayed image It is important to crop so that you can see the right edge of the gel holding area of the platform in order to maximize the area available for cutting Serial Port In the dialog box select the communications port to the spot cutter b
365. o this select Edit Calstrip Record from the Edit gt Calibrate Gels submenu The Calstrip Record dialog box will open fh Calstrip Record Editor x Name noname Description e Silver Stain 7 Fluorography 7 Syber Fluorescent Stain Detection Coomassie Stain Autoradiography Gelcode Stain A E ee Isotope Counting date dd mmm yyy s E a ge Cf DPM OD PSL Other Units EPM HALO RD INT ODu RDu ENT Number of segments O Segment values in quantitycm 2 mH mn fF rn FP FR o to FR mn Pa Pi Load Reset Delete Save Cancel Fig L 2 Calstrip Record Editor dialog box L 2 Appendix L Techniques Enter a title for the data record next to the Name prompt You can enter descriptive information about the calstrip in the Description box Next select the Detection method stain that was used to make the calstrip For Fluorography and Autoradiography detection methods select the Isotope used Also enter the date the calstrip was made next to the Counting date prompt The format in which the date should be entered is dd mmm yyyy e g 01 Nov 1993 or 01 11 1993 The first three letters of the name of the month or the number of the month may be entered Specify the units for the quantitative values by clicking on one of the buttons next to the Units prompt or typing the units in the text box next to the Other button Finally enter the Segment values for each segment of the c
366. oftware If you have Internet access please send the form directly to Bio Rad by clicking on the Submit via Internet button This will immediately update your registration If you do not have Internet access you can print out this form and send it to Bio Rad NOTE There will be a delay while we update pour registration You can either 1 Fax the form to Bio Rad at 1 510 741 5885 We will then e mail or fax you your passsword 2 Call 1 800 424 6723 x2601 inside U S or 1 510 741 2601 Intl and ask for Software Registration 3 E mail the contents of this form to LSG Software Registration Bio Rad com For network license registrations or software upgrade registrations please submit your request for a new password by email only Include your system ID purchase order number and the software catalogue number purchased along with the standard registration info in pour message text Software User Company or Institution Purchase Information Software and System Dr Mr Ms First Name NAAA EA Last Name Phone 0 A Emil fe Submit via Intemet E Print EM Close Fig 1 10 Software License Registration Form Fill out the information in the Software License Registration Form Be sure to enter your purchase order number and software serial number under the Purchase Information tab when registering Registering by Internet If you have Internet access from your computer click on the Submit via Internet
367. ogram on the Macintosh is 128 MB If the total RAM in your Macintosh is 128 MB or less you should reduce the amount of memory assigned to the program to 10 MB less than your total RAM With the application icon selected go to File gt Get Info in your Finder to reduce the memory requirements for the application See your Macintosh computer documentation for details 1 8 Introduction 1 5 Installation Windows Note Windows NT and 2000 users You must be a member of the Administrators group to install Discovery Series software After installation members of the Users group must have write access to the Discovery Series folder to use the software Insert the Discovery Series CD ROM into your computer The installer will start automatically If the CD does not auto start use Windows Explorer to open the root directory on the CD ROM and double click on the Setup exe file The installer program will guide you through the installation The installer will create a default directory under Program Files on your computer called Bio Rad The Discovery Series you can select your own directory if you wish The application program will be placed in the Bin folder inside the Discovery Series folder Additional directories for storing user profiles and sample images will also be created The installer will place an application icon on your desktop and create a Discovery Series folder under Programs on your Windows Start menu After install
368. olypeptide 4 precursor 13 81 05 0 0 0 0 0 00 Citrate proton symporter 14 75 41 0 0 0 0 0 00 URE1_MYCTU 18 63 95 0 0 0 0 0 00 Hypothetical protein ybiP 18 60 91 0 0 0 0 0 00 Coagulation factor IX factor X binding protein B chain prec 5 74 66 0 0 0 0 0 00 C4 dicarboxylate transport protein 13 70 87 0 0 0 0 0 00 Fiber protein 33 59 73 0 0 0 0 0 00 PCCS_MYCTU 20 77 74 0 0 0 0 0 00 7 Apply t Close 2 Help Fig N 13 Import Mass spec results The MatchSet is set from the one that is open when you invoke the command You must first select the instrument type used for the identification You then select the cut list submitted for identification Once this is done you can select the mass spec run for that cut list The identifications table 1s identical whether you are using PLGS or Mascot with the exception that identifications derived from using Mascot do not include MW or PI values If the identifcations have not been written to WorksBase no runs will be reported N 21 PDQuest User Guide Select the type of score you would like to see Micromass has the two forms shown The MOWSE score is selected by default most others have only the MOWSE score Select a run Once a mass spec run has been selected the samples included in the run are shown in the list on the right This will correspond to the wells on the MALDI plate containing samples from the specified cut list of the specified MatchSet Once you have selected a
369. on Model ChemiDoc XES f Referenced None Reset Referenced Camera Serial Number BR SIHL Save Auto Save After Scan Imaging rea CM Width 13 00 Height 10 30 a Reminder x Show Light Warning Display x Suto scale Transform After Acquisition ge eee Auto Exposure Threshold ow Filter warmiri x 2 Saturated Pixels lo 0D Cancel OF Fig C 11 Available options in the ChemiDoc XRS acquisition window Click on OK to implement any changes you make in this box C 5 a Imaging Area These fields are used to specify the size of your imaging area in centimeters which in turn determines the size of the pixels in your image 1 e resolution When you adjust one imaging area dimension the other dimension will change to maintain the aspect ratio of the camera lens Note Your imaging area settings must be correct if you want to do 1 1 printing These are also important if you are comparing the size of objects e g using the Volume Tools between images C 5 b Reminder Show Light Warning When this checkbox is selected the software will warn you to turn off your transilluminator light when you exit the ChemiDoc XRS acquisition window or when your system is idle for more than 5 minutes Appendix C ChemiDoc XRS Note If you are performing experiments that are longer than 5 minutes e g chemiluminescence this should be deselected Show Filter Warn
370. on Adjusting the Gamma slider handle changes the light and dark contrast nonlinearly Highlight Saturated Pixels When this box is checked any saturated pixels in the image will appear highlighted in red in the scan window and in the pop up image window To view hide saturated pixels in the pop up image window use the Image gt Transform command Invert Display This checkbox will switch light spots on a dark background to dark spots on a light background and visa versa This will only affect how the image is displayed on the screen not the actual image data Auto scale Clicking on Auto scale will adjust your displayed image automatically The lightest part of the image will be set to the minimum intensity e g white and the darkest will be set to the maximum intensity e g black You can then fine tune the display using the High Low and Gamma sliders described below Reset Reset will return the image to its original unmodified appearance A 6 Step V Analysis The Analysis step of the Gel Doc acquisition window allows you to add annotations and analyze the newly acquired image PDQuest User Guide Annotate Clicking on Annotate will open a separate image window displaying the captured image The default name for the image will include the date time and user if known The Text Overlay toolbar will also pop up to allow you to annotate your image The image will not be saved until you select Save or Sa
371. on higher level MatchSets 6 31 PDQuest User Guide With your higher level MatchSet open select Transfer MrpIs from the Analyze gt MrpI Standards submenu In the first pop up box select the MatchSet member you want to copy from in the second box select the member you want to copy to You will be asked to confirm the transfer Repeat the procedure to apply the values to all the higher level MatchSet members 6 9 Standard Spot Numbers Standard spot SSP numbers are unique numbers assigned to each spot in the MatchSet Master In general spots in the lower left region of the image have low SSP numbers while spots in the upper right region have high SSP numbers Note SSP numbers are increasingly important when you begin comparing spots between different MatchSets using higher level MatchSets SSP numbers are automatically assigned when you create the MatchSet Master To create SSP numbers PDQuest first divides the Master into rows and columns placed so that there are approximately the same number of spots in each section and that no section has over 100 spots The first two digits of a spot s SSP number correspond to its section s X and Y coordinates Spots within a section are numbered sequentially Displaying SSP Numbers The spot number tools are located on the Analyze gt Spot Numbers submenu 6 32 Analysis Tools Analyze Identify Report Y Replicate Groups a Group Consensus Describe Samples a Mormaliz
372. on which gel s to show the spots and what information to show on the gel s When you open the Analysis set Template whatever analysis sets you have selected in the ASM become inactive To view these sets in the Analysis set Template check the box labeled Display Selected sets 7 1 h Matching Analysis Sets Matching Analysis Sets are composed of spots either unique to one member or are present in all members To create a matching analysis set select matching from the Select Set Type dialog 7 20 Analysis Sets and Annotations Ll Analysis Set Template Matchset HalfStock o xj Mame Mat ch Description x Mark spots xe Operand Member hatz Display selected sets Mark spots on e Master gel Method Spots matched to ever member Operand gels f All gels f Spots unique to member Show Window labels _ 55F number Format Spot count o uf Save smal 2 Help Fig 7 9 Matching Analysis Set There are two types of Matching analysis sets The first option Spots matched to every member finds spots that exist in all members of the MatchSet For obvious reasons this option does not allow you to select a member If you select Spots unique to member the Member field becomes available Select the member from which you wish to identify spots This option will create an analysis set of all spots that are not matched to any other member in the MatchSet Click Save
373. on will allow you to select images from the disk whether or not they are open The Remove button will remove a gel from the list The Master button will designate the selected gel as the MatchSet master image This gel will be used as the starting point for constructing the eventual Master image See Section 5 7 Adding Spots to the Master for more details on constructing the Master image 4 10 Detecting and Editing Spots As indicated on the dialog you can set the order of the members by dragging them in the list This order will be used when displaying the images as well as plotting bar graphs Step 2 Detect Spots on Gels This will autmotically process all the selected images If you have processed similar images in the past you may select the same parameter set with the drop down list button If you can not use an existing parameter set press the Edit button to launch the Spot Detection Wizard and create a new parameter set Step 3 Create MatchSet Follow the same procedure as you did when you created a New MatchSet to enter the Name Description Comments and Member Sharing To specify the directory in which the MatchSet will be stored click Change Note Note If you want to detect spots without creating a MatchSet check the Disable this step checkbox at the bottom of Step 3 Step 4 Match Gels This is similar to Step 2 in that you may select existing parameters for matching or you may create new ones
374. onal to the highest bar Beneath the maximum quantitation the normalization units are displayed Normalization is a process by which quantitative data from different gels are adjusted so that you can compare different samples to one another for more information on normalization see section 6 6 If a spot is saturated the bar representing it on the histogram will be shaded red instead of black If replicate groups are defined for the MatchSet see section 6 4 you can look at the quantitation of individual spots or the average data of duplicate gels You can specify which way you want to review spot quantitation by selecting choosing a configuration in the Configure Graphs dialog See Section 11 1 for further information If you specify All Replicate Groups the bars on the histograms will display not only the maximum spot quantitation for each replicate group but also the average spot quantitation and the standard deviation Each bar represents one replicate group The order of the bars corresponds to the order in which the replicate groups were made See Section 11 1 Configure Graphs for more information PDQuest User Guide Maximum Quantity Standard Deviation Mimimum Quantity SSFP S205 Fig 11 7 Sample histogram with Replicate Group Quantitation 11 2 6 Types of Graphs Spot Review Tool Spot Review Tool on the Analyze menu section 6 1 displays a page of graphs alongside the MatchSet You can use this tool inte
375. one treatments Since protein structure and function are the direct result of gene expression the loss or change of a protein as detected by a Raw 2 D gel can be extrapolated back to events occurring at the DNA level Many questions encountered when genes are inserted and expressed in bacteria yeast and other cell types can be answered with Raw 2 D analysis Is the cell making the protein Is the cell s progeny making the protein Is the protein being made but not secreted Have mutations occurred Medical Applications Raw 2 D gels can also have important applications in medical research For example this technique can be used to verify the presence of specific protein markers that are linked to genetic diseases and disease states Used in conjunction with other tests Raw 2 D gels can be part of medical screening procedures associated with mutations and teratology linked to genetic damage Growth factors are being studied for their role in the regulation of cell growth Raw 2 D gels can be used to evaluate quantitative and qualitative changes in cellular proteins in response to growth factor stimulation Raw 2 D gels allow the visualization of proteins whose expression is altered as the result of cell transformation introducing oncogenes into the host genome Assessment of phosphorylation sulfation or other secondary modifications could reveal functional protein pathways affected by oncogene expression This information could contribut
376. ool in Step 1 to change your faint spot selection or you can increase the Sensitivity setting by typing a number directly in the field next to the Find Spot Centers button or clicking on the adjustment arrows Higher sensitivity will detect more spots If image noise has been detected as spots decrease the sensitivity or perform additional speckle removal PDQuest User Guide Min Peak If increasing the Sensitivity setting or selecting a new Faint Spot still fails to find some faint spots you can lower the Min Peak value This is the minimum peak intensity that a spot must have to be identified Again image noise may be detected as spots if the Min Peak is too low Size Scale The Size Scale field helps distinguish trends in signal intensity spots from random intensity fluctuations noise It uses the size of objects in the image in pixels to determine the nature of those objects The initial size scale setting is based on the Small Spot you selected in Step 1 If a gel image has noticeable noise features a larger size scale may be preferable If smoothing has adequately removed noise small values are preferred so that small spots will be detected You can enter odd values from 3 to 9 in the field or adjust the value using the arrow buttons Gaussian Model During Test If the Gaussian Model During Test checkbox is selected two dimensional Gaussian fitting will be performed on all spots when you find spot centers It will take
377. or each gel in the table at the bottom of the dialog box This value will be used as the denominator in the normalization formula 6 27 PDQuest User Guide This model is useful if you want to correct for known variations in samples across gels e g the amount of cells that went into an experiment the amount of extract loaded the length of time the images were detected etc Counts Loaded from Gel Record In this method the raw quantity of each spot in a member gel is divided by the total counts loaded into that gel as specified in the Gel Record when you performed calibration Note This method is only available if you have performed calibration on your MatchSet gels See Appendix J The counts loaded in each gel are listed in the table at the bottom of the Normalization dialog this value will be used as the denominator in the normalization formula Select the Counts Loaded from Gel Record option button to activate this normalization method This method assumes that few protein spots change within the experiment and that the changes average out across the whole gel This model can be useful if you want to correct for known variations in the amount of sample loaded across gels as measured prior to loading with a scintillation counter If you select this method you can compensate for pipetting errors in all MatchSet members by selecting the Pipetting Error Compensation checkbox Pipetting Error Compensation When this ch
378. or further analysis using spreadsheet programs Alternatively you can export the MatchSet to the Clipboard With the MatchSet loaded select Export MatchSet from the File gt Export submenu 2 28 General Operation 1 Export Matchset Matchset masslynx test Fig 2 19 Exporting MatchSet data In the dialog you can export data for each gel in a MatchSet Spot Data by Gel or for each replicate group in the MatchSet Spot Data by Group Under Spot Data by Gel select the data to export for each spot position quantity quality Gaussian size normalized quantity and or peak value 2 29 PDQuest User Guide The quantities of saturated spots can be estimated reported as the value 3 0 or reported in some other way that you specify If you opt for the latter click the Other button and enter the value that should be reported If a spot is unmatched in one or more members of a MatchSet it can be estimated as the value 1 0 or as a value that you enter in the text box next to the Other button For each spot you can include the SSP number DSN if designated replicate group ID and or MrpI Peak value if calculated by selecting the appropriate checkboxes You can export annotations in all categories or a specified category If you select Specified select the category from the pulldown list You can export all analysis sets or a specified analysis set To specify an analysis set select one from the pulldown list
379. or multiple spots by clicking on the appropriate button under Display combined spots Note that valid spots are spots that have not been cancelled If you elect to display combined spots they will appear in the Gaussian image as they do in the Filtered image although the quantitation will be combined In Windows Jump Cursor on Alert will automatically jump the cursor to the OK button when an alert box pops up Deselect the checkbox to turn off this feature By default a subwindow is selected when you move your cursor over it If you would rather click a subwindow to select it select Click to Focus Subwindow Note Click to Focus Subwindow will change the behavior of mouse assignable tools Some commands that were previously mouse assignable will be performed immediately on the active subwindow other commands will require you to click once in a subwindow to select it then click or drag again to perform the action Auto All Same duplicates the zoom and positioning commands made in one subwindow in all displayed subwindows For example with Auto All Same selected Zoom In on one subwindow the other open subwindows will also zoom in The Save subwindow configuration saves subwindows in the last order displayed as well as the last image type displayed which overrides the Default type selection If Bold Match Symbols is selected landmarks manual matches added spots etc will appear larger and bolder on the screen If Fille
380. order Move displayed image tl Flag spots Sl Save flagged spot set a Clear spat flags Flagged spot count s vy Done 2 Help Fig 6 4 Image Stack Tool PDQuest User Guide In the stack tool window all available gels are listed in the Select gels to display field To select an image to display click on a gel name The name is highlighted with an arrow and the image appears in the window Click on another gel name to display that image Using the Playback controls you can step forward or backward through the images in the stack tool First select some or all of the gel names using standard SFT click or CNTRL click key commands When multiple names are selected the back and forward Step arrow buttons become active Click the arrow buttons to scroll through the list of selected gels Your current gel selection is highlighted with an arrow in the gel display window Alternatively click the Auto checkbox next to the arrow buttons to begin automatically scrolling through the list You can adjust the auto scroll speed using the Slow Fast slider To stop auto scrolling deselect the checkbox You can rearrange the order of the images with the image order arrows Buttons for various viewing tools are included in the stack tool These commands will change the display of all the images in the stacker at once e g zooming in on one image will magnify the same relative area in all the images With the image flagging tools it is
381. ording to the following formula Specular OD 1 4 Diffuse OD Appendix F GS 800 F 5 6 Other Calibration Settings After you have entered the step tablet values you can immediately calibrate by clicking on the Calibrate Now button in the Densitometer Options dialog box You can also specify how often you want the GS 800 to automatically recalibrate Either click on the Calibrate Before Every Scan checkbox or specify a period between automatic recalibrations in minutes in the Recalibration Interval field Note The scanner will automatically recalibrate each time you change your filter or your reflective transmissive setting If you select a different application with the same filter and light settings it will not auto recalibrate To print out a calibration report each time the densitometer calibrates click on the Calibration Report checkbox F 6 Acquire the Image Note Before scanning in transmissive mode make sure the white balance region of the scanning area is not covered or obstructed in any way To begin to scan click on the Acquire button The scanned image will begin to appear in the scanning window line by line To interrupt a scan click on the Stop button A message will ask you to confirm the interrupt and then you will be asked if you want to keep the partial scan This feature is useful if you overestimated the size of the area you selected After the scan is complete a window will open displaying the
382. ordingly and then subtracted You can change the default reference dark exposure time using the Reset Reference button see below If you deselect the Referenced button and then reselect it the old reference dark exposure will still be available Note Separate reference dark exposures will be taken for High Resolution mode and High Sensitivity mode Once you have created a reference dark in each of these modes each reference dark will be used according to the mode you are in Reset Reference If you would like a reference dark with an exposure time that more closely matches that of your typical scans click on the Reset Reference button Reset Reference Dark Image Options Dark Exposure Time 30 oof Seconds OF Cancel Fig G 10 Reset Reference Dark pop up box PDQuest User Guide A pop up box will prompt you to enter a new reference dark exposure time in seconds Click on OK to implement your change The new reference dark will be created when you acquire your next image Note Because of the high sensitivity of the CCD fluctuations in background radiation and or temperature in the room can affect the level of dark count If you feel that radiation temperature conditions have changed in the room since your last reference dark was created use the Reset Reference button to delete your old reference and create a new one under current conditions None If you do not want to perform dark subtraction select None No da
383. ort switching gels in the spot cutter Check the Include Spot Quality box to consider spot quality when auto selecting spots When this checkbox is selected a well resolved spot of sufficient quantity will be favored over a less well resolved spot of higher quantity within the specified tolerance ratio If after analyzing the cut selections you wish to add or remove cuts click on the cells to add or remove them Note that this will automatically switch the selection mode to Manual To return to the Auto select mode click the option again A dialog opens asking you to confirm overwriting your manual edits Single gel To make all the cuts from the same gel select the Single Gel option button Then select the gel name from the pull down box All the cut requests listed in the table will be located in that gel 9 6 Integrated Excision Tool If you wish to add or remove cuts click on the cells to add or remove them Note that this will automatically switch the selection mode to Manual To return to the Single gel mode click the option again A dialog opens asking you to confirm overwriting your manual edits single group To excise all the occurrences of spots across all the gels of a specific replicate group select the Single group option button Then select the group name from the pull down box The cut requests listed in the table will be made from every gel belonging to that group Multiple gel cuts of the same spots are placed
384. ory However each annotation entry must be placed in a category and have at least one associated spot 7 23 PDQuest User Guide 7 2 0 Annotation Categories Annotations are organized in categories Some examples of annotation categories are protein name amino acid composition binding properties translational regulation etc You first select the category then enter the annotation A single spot may be annotated in multiple categories depending on the amount and type of information you know about it Most categories contain simple text annotations Specialized categories can be used to link spots to Internet protein databases or open files in other applications PDQuest includes a number of standard categories To add these to the Select Category list click the Std button below the list The standard categories will appear in the list Creating and Editing Categories To create a new category click the Add button below the Select Category list hi Add Category protname Description Type of entry Text En ALA ord Link name For the Link URL please enter the UAL of the database Including the http 2 fe UAL arg Including any arguments with a Zs where the individual spot annotations will go Filename Link URL Paces ff yf Done X Cancel Z Help Fig 7 11 Adding an URL category In the dialog box enter a Name for the category and any Description you want to include If this category
385. ot Tools submenu The dialog box will open 7 34 Analysis Sets and Annotations i Create Annotation from Analysis Set msauD0031 Step 1 Select Analysis Set new Spot count a Step 2 Select Create Annotation Category eytoskelprot Spot count 33 Create category Step 3 Enter Text for Spots in Set Add Replace Update Cancel Help Fig 7 18 Create Annotation from Analysis Set dialog box In Step 1 select the analysis set from the pulldown menu The number of spots in the set will be displayed in the Spot Count field In Step 2 select an existing annotation category from the pulldown list or create a new one by clicking on the Create Category button See Annotation Categories for more information on creating categories The initial spot count in the category will be displayed in the Spot Count field In Step 3 enter the annotation for the spots in the set Note that this annotation will be applied to all the spots in the analysis set If you are creating an annotation in a specialized category 1 e a URL or file name category make sure the entry is in the appropriate format When you have entered the annotation click the Apply Entry button An alert box will notify you how many spots were annotated in the category 7 35 PDQuest User Guide 7 9 Printing Annotations The functions for printing spot annotations are located on the Print Annotations submenu of the Analysis gt Mo
386. ot number Copy the portion of the name that coincides with the Search title and paste it into the Search title field of the Peptide Mass Fingerprint Search dialog e g the entire data file name minus the _mass txt If you use Mascot Daemon to run your search instead of the browser it automatically formats the search title for you based on the parameters set in the Daemon See Mascot documentation for using Mascot Daemon 10 10 Mass Spectrometry Analysis Highlight the desired search and the list of hits automatically appear in the Search results field Click Mascot Search Results to open theMascot browser and view the Mascort results Search Results The Search results field lists all the hits from the selected search or searches in step 2 To reduce the number of hits enter a maximum number of hits to return To apply a hit to a spot highlight the desired hit and click Apply Based on the selections you have checked in the Options dialog PDQuest automatically imports the data as annotations for the selected spot When you have finished applying hits to spots click Close For information on viewing imported data see Section 7 2 Annotation tool 10 5 a Options The top portion of the Mascot Import Options dialog allows you to select the columns you wish to appear in the Select Search field Note Selecting too many columns to view may expand the dialog beyond the display area Select only those columns necessary for your pur
387. ou use the slider bars The difference 7 10 Analysis Sets and Annotations here is you are given a view of which spots will be included or excluded from the analysis set Do you want to look for a protein whose quantitation went up under the experimental conditions as compared to the control If so click the Above Upper limit button A represents all matched spots in sample A and B represents the corresponding matched spots in sample B Comparisons are made using spot quantity only Positional comparisons etc are not possible The comparison is performed as follows A threshold value is established by multiplying and or dividing A by the factor you specify B is then compared to the threshold For example e Ifyou ask to create a set by clicking on the Above upper limit button and enter a factor of 2 0 the set will include spots whose quantity in B is at least twice that of the corresponding spot in A e If you click on the Above Upper Limit button and enter a factor of 0 5 the set will include spots whose quantity in B is greater than half the quantity of the corresponding spot in A e Clicking on the Below Lower Limit button and entering a factor of 0 5 2 0 fold will find spots whose quantity in B is less than half the quantity of the corresponding spot in A e Creating a set by clicking on Below lower limit and entering a factor of 0 33 3 0 fold finds all the spots whose quantity in B is less than one third the quant
388. ouch but do not overlap Step 2 Specify Cuts F Add cut T Remove cut X Multiple cuts spot Cuts o Max cuts spot fs Excisions o x Snap to peak Plates o Step 3 Set Cutter Options X Spin when cutting _ Cut longer x Spin when ejecting X Double eject No spin when lifting JBacked gel Tip motion range 2 z0 Gel defaults PYDF defaults Fig 8 9 Multiple cuts to be placed in different wells Note Overlapping circles will result in incomplete cuts that may not be fully extracted by the spot cutter To place multiple cuts of the same spot in the same microtiter plate well select the Multiple Cuts Spot checkbox and enter the maximum number of cuts per spot in the Max Cuts Spot field Click the Add Cut button then drag a box over the spot Multiple cutting circles will appear grouped inside a red box All these cuts will be placed in the same well 8 14 Basic Excision Tool Step 2 Specify Cuts TA Add cut We Remove cut x Multiple cuts spot Cuts ca Max cuts spot cm Excisions oa Xx Snap to peak Plates oui Step 3 Set Cutter Options x Spin when cutting 7 Cut longer x Spin when ejecting X Double eject O No spin when lifting Backed gel Tip motion range lz ae Gel defaults PDF defaults 1 404 Fig 8 10 Multiple cuts to be placed in the same well Note Ifthe box you create contains too few cutting circles simply drag over the a
389. over a period of time The effect is analogous to exposing photographic film to light over a period of time Auto Expose Auto Expose will take an exposure whose time length is determined by the number of saturated pixels in the image This is useful if you are uncertain of the optimal exposure length PDQuest User Guide Note If you know the approximate exposure time you want 3 seconds you can skip this step and go directly to Manual Expose Click on the Auto Expose button to cancel Live Focus mode and begin an automatic exposure The Auto Expose button will appear selected throughout the exposure During the auto exposure the image is continuously integrated on the camera CCD until it reaches a certain percentage of saturated pixels This percentage is set in the Options dialog box Default 0 15 percent See section A 9 Options Step IIl Acquire Image Click on Auto Expose button appears depressed Exposure Time sec Oo Freeze Fig A 4 Auto Expose Can see Exposure Time automatically changing Once an image has reached the specified percent of saturated pixels it is captured and displayed in the video display window Auto Expose is automatically deactivated the exposure time appears active in the Exposure Time field and Manual Expose is activated Note If you are having difficulty auto exposing your sample you can use Manual Expose to adjust your exposure time directly Most applications o
390. ower left Sipate x Cutter x Camera Focus cutter camera Move head to upper right Set cutting Itip height Fig 8 2 spot cutter setup 8 1 a Focus Cutter Camera Note If you are using a lens filter make sure that it is installed before focusing If you add or remove the lens filter after focusing you will need to refocus The Focus Cutter Camera dialog box controls the camera exposure setting Focusing and aligning the camera are described in the Proteome Works spot cutter hardware manual e Click the Focus Cutter Camera button to open the dialog The platform light a on the spot cutter will turn on and the camera will focus on a small region at the center of the gel or membrane and begin taking continuous exposures The exposures will appear in the Focus window at the rate of about 2 per second At the top of the dialog click Auto Expose to reset the exposure time of the camera The Time field shows the time of each exposure in milliseconds The Ceiling field shows the highest pixel value in the focused region The Levels field shows the levels of gray scale in the image All of these settings are automatically determined by the software based on the position and aperture of the camera lens Manually adjust the camera lens and position as described in the hardware manual to optimize the image When you are satisfied with the image and the levels of gray scale click Stop to close the Focus dialog 8 1 b Set Cutting
391. ox You cannot save preview scans If you want to stop a preview scan that is in progress click on the Stop button H 5 Acquire the Image The Fluor S MAX gives you the option of simply acquiring and displaying a fully exposed image or preserving intermediate exposures Acquire To acquire and display a fully exposed image click on the Acquire button An exposure will be taken based on the time selected in Step III This is appropriate for most short exposures The progress of each exposure will be displayed in the Exposure Status bar at the bottom of the acquisition window Exposure Status A JAcquiring Image Fig H 9 Exposure Status bar when acquiring an image Depending on which dark subtraction type you have selected a dark count may be acquired immediately following image acquisition See Dark Subtraction Type under Options below If you want to stop a scan that is in progress click on the Stop button The acquisition will be terminated H 10 Appendix H Fluor S MAX After an image has been acquired a separate window will pop up containing the new image The window will have a default file name that includes the date time and user if known To save the image select Save or Save As from the File menu You can then analyze the image using the standard analysis functions Live Acquire Live Acquire allows you to view and preserve intermediate exposures leading up to a full exposure This is useful for
392. pear in this window PDQuest User Guide The control panel has been arranged from top to bottom to guide you through the acquisition procedure There are five basic steps to scanning an image using the GS 710 l Ze E 2 Select the application Select the scan area Select the resolution of the scan Calibrate the instrument This is automatic after you enter the step tablet values before you first scan after installation Acquire the image Step I Select Application To set the parameters for your particular scan you can l 2 Select from a list of possible applications or Choose your own filter and light source settings Selecting from the List of Applications When you select from the list of applications the software automatically sets the appropriate filter s and other parameters for that particular application To select from the list of applications click on the Select button under Select Application Appendix E GS 710 ray film Coomassie blue Silver stain Copper stain Zinc stain Stain all Fig E 3 Example of the application tree in the GS 710 dialog box The applications are listed in a tree that expands from left to right First you select the category of your application then you select your particular application To exit the tree without selecting press the ESC key Gray file X ray film Blue film Linear Coomassie Blue Silver stain Gel Copper stain
393. pendix B ChemiDOC sissa naa aE B 1 Appendix C ChemiDOC XRS siiis ni ERa A C 1 Appendix D GS 700 Imaging Densitometer ccccssseeeesseeesenseeeeeneees D 1 vi Contents Appendix E GS 710 Imaging Densitometerr cccsseeseeseeeenseeeeneeeeees E 1 Appendix F GS 800 Imaging Densitometer cccccsssseeceeeseeseesseeseeees F 1 Appendix G Fluor S Multilmager sevcessiacicssccctsancctcneresscedenennstssewedeccscdsaeecenicess G 1 Appendix H Fluor S MAX MultilMager cccccsseeceeseeceseesseeeeeeeeeeseeneenes H 1 Appendix Personal Molecular Imager FX cccsssseseseeeeseeeeseeeeeeeenseeeaes l 1 Appendix J Molecular Imager FX Family FX Pro FX Pro Plus and Molecular Ea J 1 Appendix K NOESADOG miii K 1 Appendix L Calibration and Merging ccccssseeesssseeeseesseeceeneeeseesneeseeaes L 1 Appendix M Cross Platform File Exchange ccccccseseeessenseeseeneeeeeeeseeeees M 1 Appendix N WOFKSDASO siesaicsreacncatnarevenauesveaaseanesciccdarscaveaiecanassivasecsawetiesdeuaaets N 1 vii PDQuest User Guide viii Preface 1 About This Document This user guide is designed to be used as a reference in your everyday use of PDQuest It provides detailed information about the tools and commands of PDQuest for the Windows and Macintosh platforms Any platform differences in procedures and commands are noted in the text This gui
394. pendix H Fluor S MAX Recommended Exposure Times and Lenses Recommended Exposure Lens amp Filter Accessories Used Fluorescence End Label Gel Colorimetric Gel White Diffusion Plate Colorimetric Blot 0 2 10 sec Zoom IR None X ray film Zoom IR White Diffusion Plate Weak Chemiluminescence 2 10 min 50 mm Chemi Tray if sample is small Strong Chemiluminescence 5 sec 1 min 50 mm Chemi Tray if sample is small For sharper focusing close the f stop down 1 2 stops from full open while focusing For chemi applications the 50mm lens is recommended Always remove the 660 filter For most applications you can select an exposure time capture an image study it then adjust the exposure time accordingly Repeat this procedure as many times as necessary to obtain a good image For chemiluminescent samples which degrade over time and emit low levels of light you can select a high exposure time initially and use Live Acquire mode to save intermediate exposures see following section Preview For shorter exposures you can use Preview to test different exposure times Click on the Preview button create a preview exposure and display it in the acquisition window PDQuest User Guide A preview scan takes only half as long to create as a real scan because the preview scan does not capture a dark image see below The progress of the exposure will be displayed in the Exposure Status bar at the bottom of the dialog b
395. ph Partial Matches Graph Partial Matches Match gt Edit Matches submenu displays histograms of each partially matched spot in the Master Select the command from the submenu and click in the Master Use Graph Advance Graph Retreat to graph different regions of spots Graph Erratic Responses Graph Erratic Responses Match gt Edit Matches submenu displays histograms of each erratic spot in the Master Select the command from the submenu click in the Master enter a fold change factor in the pop up box and click Apply to display the graphs Use Graph Advance Graph Retreat to graph different regions of spots Graph Advance Graph Retreat If you are displaying Margin Graphs Column Graphs or graphing spots using the Graph Partial Matches or Graph Analysis Set commands you will need to shift the display to graph spots in different regions of the image The commands for doing this are located on the Reports menu or the Graph toolbar Graph Advance SFT PAGE DOWN shifts the column of spots being graphed to the right Graph Retreat SFT PAGE UP shifts the column of spots being graphed to the left For Margin Graphs these commands flip through the graph pages 11 3 A B Overlay The A B Overlay function is used on MatchSet Masters to highlight and compare the spots that appear in two different MatchSet members PDQuest User Guide Select A B Overlay from the Reports menu and click the Master of the MatchSet in which you want to perform
396. phase will make lower confidence matches The behavior is similar to lowering the precision in the primary phase but the matches are displayed in a different color This allows you to keep the precision high in the primary phase and review only the extended matches The extended match phase allows you to select several methods one of which is the classic method This will use the same 9 17 PDQuest User Guide algorithm PDQuest has used in previous versions If you select this method the primary phase can be thought of as auto landmarking Extended matches are shown in blue Getting started First you must create a MatchSet and designate a Master image Once this is done select Match gt Automated Matching If you simply want to match all the spots using the default settings press the Match All button then the Done button If you want to explore the match setting select a member using the Members pull down button Then use the sliders to change the settings and press the Match button You will see the number of primary matches and the number of extended matches on the dialog You will probably want to configure the MatchSet window to display only the master and the member you are matching You can do this with theWindow gt Configure Sub windows command Inspect the matches for accuracy and completeness Once you have obtained the best settings for your images press Match All to apply those settings to all members and match
397. plates high density arrays samples with very closely spaced bands Files scanned at 50 micrometers can be very large e 100 micrometer resolution should be used for typical gels and arrays e 200 micrometer resolution is useful for gels with large bands and dot blots e 800 micrometer resolution should be reserved for very large objects such as CAT assays PDQuest User Guide File Size of Images Image File Size below Select Resolution shows the size of the scan file you are about to create If you do not have enough computer memory for the specified file size an error message will appear when you attempt to scan If this happens select a lower resolution or decrease the size of the area to be scanned Macintosh users can also increase the application memory partition See your Macintosh computer documentation for guidance 1 4 Acquire the Image Once you have selected your scan area and resolution you ready to acquire an image Click on the Acquire button There may be a short delay while the image laser warms up then the scanned image will begin to appear in the scanning window line by line To interrupt a scan click on the Stop button A message will ask you to confirm the interrupt and then you will be asked if you want to keep the partial scan This feature is useful if you overestimated the size of the area you selected Note Ifthe image you are scanning has more than 10 saturated pixels you will receive a warning
398. poses The lower portion of the Options dialog allows you to select the types of hit data you wish to import 10 11 PDQuest User Guide 10 12 11 Graphs and Reports This chapter describes the graphs and reports found in PDQuest While most of the graphs and reports described in this section are available from the Report menu Spot review is found in the Analyze menu and Graph Partial Matches and Graph Erratic Responses are both located in the Edit Matches submenu of the Match menu Report Window Help Graph Tools xj Configure Graphs me AR AR T Fop up Graph FIO Margin Graph Shitt F10 Column Graph Graph Advance Shift Page Down Graph Retreat Shift Page Up Spot Review Tool A B Overlay Quantity Table Report Quantity Graph Report Master Image Report Scatter Plot Report Fig 11 1 Reports menu and Graph Toolbar 11 1 Configure Graphs The Configure Graphs tool of PDQuest allows you to configure the way graphs are displayed The Configure Graphs tool includes two configurations all gels and replicate groups with the ability for you to create more Open the Configure Graphs tool by selecting Report gt Configure Graphs PDQuest User Guide Ll Configure Graphs HalfStock E 08 x 411 Gels All Replicate Groups J Apply Add Edit Delete Ma Help Fig 11 2 Configure Graphs tool To select a graph configuration highlight the desired configuration and click Apply The
399. pots from the Gaussian Fw image select Remove Spot from the Spots menu or toolbar then click individual spots or drag a box around a group of spots in the Filtered image The spot s will disappear from the Gaussian image and the spot crosshair s will disappear from the Filtered image 4 17 PDQuest User Guide Note Itis also possible to add spots on Raw 2 D scans when the Filtered and the Gaussian images are loaded Problem Too many spot centers Result Too many Zo appear in identified in the Filtered image Gaussian image Solution Use Remove Spot then Result Correct number of spots in the Add Spot to reposition spot centers Gaussian image Fig 4 11 Removing incorrectly identified spots in a cluster 4 6 Spot Boundary Tools If a spot is saturated irregularly shaped or otherwise of poor quality then Gaussian modeling will be unable to accurately determine quantity In these cases you can manually define the spot in the Filtered or Raw 2 D image using the spot boundary tools Note For most spots Gaussian modeling results in more accurate quantitation than defining the spot boundary When you define a spot boundary the region inside the boundary is identified as the spot and is added to the Gaussian image Spot quantity is calculated by summing the intensities of the pixels inside the defined boundary You can define the boundary of any spot in a Filtered or Raw 2 D image 4 18 Detecting and Editing Spots
400. print out a scatter plot report for all the member pairs of a MatchSet A scatter plot shows the relatedness of two different gels that have been matched in a MatchSet Note To display a scatter plot for any two gels in a MatchSet use the Scatter Plot Tool in the Analysis menu section 6 3 Scatter Plot Tool 11 14 Graphs and Reports il Scatter Plot Report HalfStock 3 Eo x MatchSet name ey Clocl eee eee ee Title A A Researcher E Institution oe am ae O Gate lt a a a nee al wf Done X Cancel E Help Fig 11 14 Scatter plot Report When you select Scatter Plot Report from the Report menu a dialog opens in which you can enter a report title researcher name and the name of the research institution Click Done to open the report viewer 11 15 PDQuest User Guide Reformat Fig 11 15 Scatter Plot Report Viewer The report viewer allows you the option of printing or reformatting the report Click the reformat report button to return to the Scatter Plot Report dialog 11 16 Appendix A Gel Doc 2000 Fig A 1 Gel Doc Before you can begin acquiring images the Gel Doc system must be properly installed and connected with the host computer See the Gel Doc hardware manual for installation startup and operating instructions PDQuest User Guide To use the Gel Doc you will need to have the Bio Rad supplied acquisition board installed in your PC or Macintosh The drivers for this board wi
401. proceed or cancel the save operation File name Last saved on Last saved by Watz v1 Raw 2 D Image Matt v1 Filtered Matt v1 Gaussian 2002 05 1 9 1 4 44 4SUPERUSER 2002 05 1 9 1 4 44 2SUPERUSER 2002 05 1 9 1 4 44 2SUPERUSER Fi Proceed 2 Cancel Fig N 10 Confirm Overwrite dialog box N 6 Automated detection and Matching in WorksBase The Automated Detection and Matching tool combines several processing steps into a single operation To select images from WorksBase for processing select Spots gt Automated Detection and Matching in WorksBase This opens the Automated Detection and Matching dialog N 15 PDQuest User Guide li Automated Detection and Matching in WorksBase ee x rStep 1 Select Gels To Analyze Step 3 Create MatchSet Add 42degreet Name MS00017 2002 04 02 Auto 42degree2 Description ER Remove 42degree3 Comments 83 Master ES Master rAssociated records Lar Program Matt s Program a Project Matts Project A gt Nay Esetiment e expt YY Count 3 i Drag items to reorder them _ Disable this step Step 2 Detect Spots on Gels Sieg A Match Gel Parameters Matts pa hd Edit Parameters Defaut Match Settings v Edit _ Disable this step Log File Name MHalsey_20020402_1634 Write dataset write dataset for Ahmed Go X Cancel 2 Help Fig N 11 Automated Detection and Matching
402. r please contact Bio Rad during US Pacific business hours Phone 1 800 424 6723 2601 in the U S Fax e mail 1 510 741 2601 Intl Ask for Software Registration 1 510 741 5885 LSG Software Registration Bio Rad com Please provide the information in the completed registration form For network license registrations or software upgrade registrations please submit your request for a new password by email only Include your system ID purchase order number and the software catalogue number purchased along with the standard registration info in pour message text Current license Single system license License expires on 24 Aug 2002 System ID CMXXXLXNE3 Password Quantity One OK Not OK Enter Cancel Fig 1 11 Enter Password screen In the Enter Password screen type in your password in the field 1 15 PDQuest User Guide Once you have typed in the correct password the OK light next to the password field will change to green and the Enter button will activate Click on Enter to run the program 1 9 Downloading from the Internet You can download a trial version of the software from Bio Rad s Web site Go to the Discovery Series download page at www bio rad com and select from the list of applications Follow the instructions to download the installer onto your computer then run the installer After installation double click on the application icon to run th
403. r Guide users you should move the lower level MatchSets to a shared file server and create it there Note Ifa higher level MatchSet and or its constituent MatchSets have been moved from their original location you can edit the higher level MatchSet file to make it openable First open the MatchSet folder using Windows Explorer or Macintosh Finder then open the descript file a text file inside the MatchSet In the descript file the path links to the lower level MatchSets are shown You can change these paths to the new location to make the higher level MatchSet openable i Create MatchSet on disk E x Name e est Auto Description Comments MatchSet level Member sharing Do not make copies refer to the originals C Level 1 members are gels Copy the originals into this matchset s data folder Higher level members are MatchSets Members Add test2 test Remove MS00002 2001 10 04 ti Master Count 3 ing items to reorder them Save X Cancel 2 Help Fig 5 21 Creating a higher level MatchSet You cannot create a Higher Level MatchSet from the Automated Detection and Matching dialog nor from the Edit MatchSet dialog 9 36 Matchsets Click Add to open the Select Member dialog where you can highlight the MatchSets you want to include Click open and they will display in the Members list Select one Matchset Master gel to use as the MatchSet M
404. r MatchSet open select Protein Probe from the Identification menu and click on a spot for which you ve imported data The Protein Probe browser will open See your Protein Probe documentation for information on how to use the browser 10 5 Import Mascot Results When importing results from Mascot PDQuest searches the searches log file to find results that apply to the current MatchSet Once the identifications have been applied the researcher can right click on a spot launching the Mascot browser with the corresponding data file Before you can import Mascot results you must map the directories where your Mascot install folder is located and enter the URL for the Mascot browser Go the Edit menu select preferences and click on the Paths tab Click browse to locate the Mascot install folder and enter an address for the browser The Import Mascot Results dialog allows you to import data from a Mascot spot identification run To import Mascot results select Identify gt Import Mascot results This opens the Import Mascot Results dialog 10 7 PDQuest User Guide fh Import Mascot Results 4500064 2002 05 01 o i GJetzon MSO000142002 04 26 selectedspots_1_PlateOO1 1 5303 beta lactamase EC 3 5 2 6 phage phi x174 31537 24 BETA LACTAMASE Cloning vector ps45 Cm 31563 25 BETA LACTAMASE PRECURSOR EC 3 5 2 6 Cloning vector pQESONST 31507 23 BETA LACTAMASE Cloning vector pM 31559 27 BETA LACTAM
405. r matching a partial plate to the load order of a digest robot Well A1 position The fields and buttons in this section are used to calibrate the position of the microplate for proper loading of samples The number fields contain the X and Y coordinates in millimeters of well Al on a plate in the spot cutter plate holder Once you have set the position of this well the cutter robot will automatically calculate the positions of the remaining wells To set the well Al position position your cursor in the number in the X or Y coordinate field click to place the cursor in the number field and press Enter on your keyboard The tip will move to that position without attempting to lower into the well If the tip is not centered over the well A1 on the microplate you will need to adjust the numbers in each field You can either type a new value directly in each field or for small adjustments use the arrow buttons to adjust the tip by fractions of a millimeter The cutting tip will move after you press Enter or when you click the arrow buttons Note that the arrow buttons are oriented as if you were facing the front of the spot cutter also if you are facing the cutter higher numbers in the first X field will move the cutting tip to the right while higher numbers in the second Y field will move the cutting tip toward the back of the instrument When you are satisfied with the position of the cutting tip click Test to lower the tip int
406. ractively with the MatchSet to review spots Pop up Graph Pop up Graph F10 shows a histogram next to a selected spot Select Pop up Graph from the menu or toolbar then click on a spot With Pop up Graph only one histogram can be displayed in a window at a time If you select another spot the first histogram disappears and a new one displays Margin Graph Margin Graph SHIFT F10 on the Reports menu and Graph toolbar will display histograms for several spots along the left and the right margins of the screen Select Margin Graph then click on several spots Their histograms will be displayed in the left and right margins The Margin Graph only applies to the MatchSet Master Depending on the size of your window you will be able to display up to 10 margin graphs at once If you keep clicking on spots after you ve displayed 10 graphs the graphs will be displayed on a new page Use the Graph Advance shift page down Graph Retreat shift page up commands to flip through the pages Graphs and Reports Column Graphs Column Graph on the Reports menu and toolbar will fill the right and left margins with histograms of spots in the Master gel Select Column Graph the spots whose graphs are displayed are all located in a narrow column of the image centered around the location of the cursor The Column Graph only applies to the MatchSet Master To change the column of spots use the Graph Advance Graph Retreat tools see below Gra
407. ransmissive scanning the scanner is calibrated against a transmissive step tablet In reflective scanning the scanner is calibrated against a reflective step tablet unless the reflective calibration strip is uncalibrated If the reflective calibration strip does not have step tablet values associated with it you can use the default values in the form In the Tablet Serial Number field you can type in the serial number for the step tablet values you are entering The reflective step tablet will probably not have a serial number Your calibration strip will have a clear plastic cover If the quantity offset value for the clear cover is included in your calibration strip package you can enter this number in the Quantity Offset field Otherwise use a value of 0 045 D 12 Appendix D GS 700 Finally enter the values in the appropriate fields under the Diffuse column After the step tablet is scanned the software will associate each density value with its corresponding segment on the step tablet The step tablet density values do not need to be entered each time you calibrate Once they have been entered and saved they will be automatically recalled when the calibration strip is scanned You only need to enter new values if you use a new step tablet When you finished filling out the Step Tablet Values form click on OK Diffuse Versus Specular O D In the step tablet form you enter O D as diffuse density and then the software
408. re Annot Tools submenu You can also print annotations from the Browse Annotations page Print Annotations One Spot Specify Categ s One Spot All Categ s Transfer Annotations Analysis Set Specity Categ s Analysis Set All Categ s All Spots Specify Categ s All Spots All Categ s Fig 7 19 Print Annotations submenu Using the commands in this submenu you can print annotations for individual spots the protein spots in an analysis set or all the spots in the MatchSet Master You can also print annotations for particular categories or all categories Printing Annotations for Individual Spots To print the annotations for a particular spot select One Spot Specify Categ s or One Spot All Categ s then click on the spot in the Master Note that Browse Annotations will also print out all the annotations for a particular spot If you select One Spot Specify Categ s a pop up list will prompt you to select annotation categories to print Once you have made your selections click Done Printing Annotations for Analysis Sets To print annotations for spots in a specific analysis set select Analysis Set Specify Categ s or Analysis Set All Categ s from the Print Annotations submenu A list of analysis sets will be displayed Click on the name of the set whose annotations you want to print If you selected Analysis Set Specify Categ s a pop up list of categories will appear Make your selection s then click
409. re Version 1 0 Save _ Auto Save After Scan White EFI Light available UV EFI Light Available Sere UW Trans Light Available Displ Imaging Area CM paii width Baz JAuto cale Transform After Acquisition Height 5 12 Cancel OF Fig K 8 Options dialog box K 6 a Dark Subtraction Type All CCD cameras accumulate electrons that produce a signal that is indistinguishable from light This dark current adds to the noise in your images particularly for long K 14 Appendix K VersaDoc exposures In most cases you will want to subtract this dark current from your images The settings for subtracting the dark current are in the Options dialog box Click on the Options button in the acquisition window to open this dialog Normal The Normal option button selects the default dark subtraction type In this mode after you acquire an image a dark image of the same exposure length will be taken and this will be subtracted from your image The progress of the dark exposure will be displayed in the Exposure Status bar following the regular image exposure In Normal mode a dark image is only acquired the first time you perform a scan with particular application and exposure settings If you perform subsequent scans with the same settings no dark exposure will be taken Reference If you do not want to perform a dark exposure with each acquisition you can take a reference dark exposu
410. re composed of spots that are present in one gel but not in another 1 e spots that have been turned on or off Quantitative Analysis Sets Are composed of spots whose quantitation has increased or decreased x fold where x is a user determined value or whose quantitation has changed above or within the fold change factor that you specify Statistical Analysis Sets Are composed of spots that are significant based on the statistical test that you select Arbitrary Analysis Sets Are composed of spots that you manually select 7 1 PDQuest User Guide Boolean Analysis Sets Are created by comparing two or more other analysis sets For example set C could be made up of those spots that are present in both set A and set B Matching Analysis Sets Are composed of spots which are either unique to one member or are present in all members Analysis Sets in Higher Level MatchSets When you create a higher level MatchSet in PDQuest analysis sets created in the members are automatically transferred as part of the higher level MatchSet In addition you can create analysis sets in the higher level MatchSet There are three types of analysis sets you can create in a higher level MatchSet Arbitrary Analysis Sets Boolean Analysis Sets and Matching Analysis Sets Note Transferred analysis sets cannot be edited 7 1 a Analysis Set Manager The Analysis Set Manager ASM is a convenient tool which allows you to crea
411. re that will be saved and subtracted from all subsequent acquisitions Click on the Referenced button to activate this feature The first time you acquire an image after selecting this option the VersaDoc will take a dark exposure that will be saved and used to subtract the dark current from all subsequent acquisitions Note The VersaDoc 1000 will take a 60 second reference dark exposure the VersaDoc 3000 and 5000 will take a 180 second reference dark exposure For image exposures that are longer or shorter than the reference dark the reference dark will be scaled accordingly and then subtracted It is recommended that the reference dark exposure time be equal to or greater than the sample exposure time You can change the default reference dark exposure time using the Reset Reference button see below If you deselect the Referenced button and then reselect it the old reference dark exposure will still be available K 15 PDQuest User Guide Separate reference dark exposures will be taken for images that have different levels of binning or gain Once you have created a reference dark for each level of binning or gain the appropriate reference dark will be used according to the settings of your selected application Reset Reference If you would like a reference dark with an exposure time that more closely matches that of your typical scans click on the Reset Reference button A pop up box will prompt you to enter a new dark e
412. re you install the VersaDoc camera interface adapter You can find the README documents in the VersaDoc Drivers folders on The Discovery Series TDS CD PDQuest User Guide The CD also contains Roper Scientific camera interface driver installation instructions for Macintosh computers See the VersaDoc hardware manual for further installation startup and operating instructions for the VersaDoc Imaging System K 1 VersaDoc Acquisition Window To acquire images using the VersaDoc go to the File menu and select VersaDoc The acquisition window for the instrument will open The acquisition window opens displaying a control panel and an image display window YersaDoc Step Select Application Y Channel 1 Channel 2 Channel 3 Channel 4 iX Enable Channel Select SYPRO Orange S520LP Uv TRANS 4x Gain 1x1 Bin Step II Position Focus Position Focus z 3 Lights ON Step Ill Set Exposure Time sec fa o etl O Preview X Flat Fielding O Acquire Optimize Exposure 3 Stop Display Options Highlight Saturated Pixels Image file size 0 50 Mb Options Help eee 5 Fig K 2 VersaDoc acquisition window When the VersaDoc window first opens no image will be displayed The control panel has been arranged from top to bottom to guide you through the acquisition procedure There are four basic steps to acquiring an image using the VersaDoc Appen
413. rea again to create a bigger box To move a group of multiple cuts with Add Cut selected position your cursor inside the center circle in the box The appearance of the cursor will change to a multidirectional arrow Drag the box to a new location and release the mouse button Removing Cuts To remove a cut circle from the image position your cursor on the spot and right click You will be prompted to confirm the deletion To remove multiple cut circles click on the Remove Cut button then click on the cut circles or drag a box around the circles Display Options Click the Well Number checkbox below the Basic Excision image window to display the well number associated with each cut request Click the Cut Position checkbox to display the position of the cut in millimeters on the gel 8 15 PDQuest User Guide 8 2 C step 3 Set Cutter Options Before beginning a cut run using the Basic Excision Tool you must select the cutter options for that run Select Spin When Cutting to make the cutting tip spin as it cuts This is recommended for all cutting including gels and blots Deselect this option if you want to test X Y positioning without cutting Spin When Ejecting will cause the cutting tip to spin as it ejects the cut into a microplate well This is only recommended for PVDF or nitrocellulose blots Cut Longer will cause the cutting tip to hold in the down position longer when making a cut Double Eject will cause
414. receive a software package and a Hardware Security Key You can then complete the registration process as described in the previous sections 1 10 PDQuest Basic In order to meet requests for additional software copies at low cost when buying an imaging system Bio Rad has provided a scaled down version of PDQuest called PDQuest Basic PDQuest Basic looks and acts exactly like the full version of PDQuest but with the advanced functions inactive grayed out The Basic version of PDQuest includes the following active functionality Image acquisition Transform Crop Flip Rotate Text Tool Basic Excision Print and Export to TIFF and Save PDQuest Basic also allows you to view previously saved data created using advanced functionality PDQuest Basic does not require a software license To activate the advanced commands of PDQuest contact Bio Rad for a valid user license 1 11 Contacting Bio Rad Bio Rad technical service hours are from 8 00 a m to 4 00 p m Pacific Standard Time in the U S Phone 800 424 6723 510 741 2612 Fax 510 741 5802 E mail LSG TechServ US O Bio Rad com For software registration PDQuest User Guide Phone 800 424 6723 in the U S 1 510 741 6996 outside the U S 2 General Operation 2 1 Graphical Interface 2 1 a Menu Bar PDQuest has a standard menu bar with pulldown menus that contain all the major features and functions available in the software i PO QUEST File Edit View I
415. recommend that you archive images on a network file server or high capacity removable disk PC The following is the recommended system configuration for installing and running on a PC Operating system Processor RAM Hard disk space Monitor Windows 98 SE Windows NT 4 0 with service pack 6 Windows 2000 Windows XP Pentium gt 333 MHz gt 128 MB or better for Gel Doc ChemiDoc ChemiDoc XRS and VersaDoc systems gt 256 MB or better for Molecular Imager FX systems Personal FX system and GS 800 densitometer 3 GB 17 monitor 1024 x 768 resolution absolutely required True color 1 7 PDQuest User Guide SCSI Printer Macintosh Required for all Bio Rad imaging devices except the Gel Doc ChemiDoc ChemiDoc XRS and VersaDoc systems Adaptec SCSI card recommended Optional The following is the recommended system configuration for installing and running on a Macintosh Operating system Processor Model RAM Hard disk space Monitor SCSI Printer System 9 2 or higher excluding Mac OS X PowerPC G3 processor or better gt 256 MB for all Bio Rad imaging systems gt 3 GB 17 monitor 1024 x 768 resolution absolutely required Millions of colors Required for all Bio Rad imaging devices except the Gel Doc ChemiDoc ChemiDoc XRS and VersaDoc systems Adaptec SCSI card recommended Optional Note The default amount of memory assigned to this pr
416. replicate groups and classes in a MatchSet or a selected analysis set in a MatchSet With a MatchSet open select Scatter Plot from the Analyze menu When the scatter plot window opens the first two gels in the MatchSet are selected by default Choose whether you want to view gels replicate groups or classes Next select different gels replicate groups or classes to compare from the pull down list buttons next to the X Axis and the Y Axis fields PDQuest User Guide i Scatter Plot HalfStock Compare e Gels Repl Gels A Anis stocki E r Asiz stock2 Classes Markers e Hold 3 fold 4told None X Regression line Corr coeff 0 939181 Slope 0 954056 Intercept 3 068563 Spot count 903 Noru units Un norm Click on a dot to center the images uf Done E Print BPrint an 1 Help Fig 6 5 Scatter Plot dialog The quantity of each spot in the first item X axis is plotted on a log scale against its quantity in the second item Y axis If the spot s quantity is the same in both its point Analysis Tools will fall on the black center line in the graph slope 1 00 If the quantities are not the same the point will fall above or below the center line The regression line generated from the plot is shown in green This is displayed when the Regression Line checkbox is selected If you select the 2 fold 3 fold or 4 fold buttons parallel red and blue lines w
417. represents the O D value for the segment Beneath each box is the number of counts associated with that segment from the Calstrip Record If you make a mistake simply redraw a new box over the old one Repeat this procedure for all the segments of the calstrip Note All segments entered in the calstrip record must be boxed starting from the left The leftmost segment corresponds to Segment 1 on the Calstrip Record Your calstrips and calstrip data should be designed so that either 1 the darkest segments are on the left and the count values decrease or 2 the lightest values are on the left and the count values increase L 7 PDQuest User Guide You can draw more boxes than the number of segments defined in the Calstrip Record However they are automatically and permanently deactivated L 1 d Plot the Calibration Curve Once you have created the sampling boxes select Plot Calib Curve to display the calibration curve A graph will be displayed that correlates the optical density usually between 0 00 and 2 00 O D with DPMs or other specified units The Y axis represents the optical density 0 00 2 00 in each sampled segment while the X axis represents the corresponding DPM or other unit per image unit area calculated from the calibration record data Fig L 4 Example of a calibration curve Appendix L Techniques Calibration Curve Problems Find errant data points and their corresponding segments using the fu
418. responding to the image being saved Note A yellow finger icon indicates a required field Name The image name is derived from the scan file and cannot be changed N 12 Appendix N WorksBase Version The version number is typically used to distinguish versions of the image resulting from smooth or crop operations Description You may enter a longer description of the image here Program This is the Research Program under which this experiment is being performed It is considered the highest unit of organization A Program can comprise many Projects Project This is the Project under which this experiment is being performed A Project can contain many experiments Experiment A set of processes designed to test a single hypothesis Sample A biological sample collected in the course of the experiment Gel A gel run from a single sample This is the 2nd dimension gel record On the form is a link to the Ist dimension gel record which may be entered there N 4 a Image Analysis Experiment Step If the checkbox Create Image analysis step located in the WorksBase Options display tab is checked PDQuest automatically creates an Experiment Step called Image Analysis that appears in WorksBase for Proteomics This saves you the trouble of manually creating the step as an experiment step is always required This option is checked by default If you do not want PDQuest to automatically create this step go to WorksBase Op
419. rk exposure will be acquired or subtracted G 6 b Live Exposure Mode Exposure Count If you are using the Live Acquire function see previous section you need to specify how many intermediate exposures you want to view save during acquisition Enter this number in the Exposure Count field The total exposure time will be divided by the number you enter in the Exposure Count field If you enter an exposure time of 10 minutes and a count of 10 you will create 10 intermediate exposures at 1 minute intervals Note Do not enter a count that will result in an intermediate exposure time that is less than the minimum exposure time for the mode you are in The minimum exposure time in trans illuminated mode is second and the minimum exposure time in epi illuminated mode is 0 1 second Example For a trans illuminated application an exposure time of 20 seconds and an exposure count of 21 would result in an error Save All Intermediate Images If Auto Save After Scan is selected see following section the Save All Intermediate Images checkbox will become active If you select this checkbox all your intermediate exposures will be saved as separate files These files will have the same G 14 Appendix G Fluor S root name appended by a number indicating the exposure sequence The final full exposure will have the root name only with no exposure number G 6 c Save Auto Save After Scan To automatically save any image you crea
420. robe to launch the Protein Probe browser for each sample Annotation Categories Created When you click Apply the following annotation categories are created and the mass spec data for each spot is entered as an annotation in the appropriate category See section 7 2 6 Annotation Categories for more information on annotation categories e Protein Name The annotation entry in this category is the name of the protein selected by you from the hit list e Accession Number The annotation entry in this category is the database number keyed to the selected protein name Note that some databases e g Swissprot include accession numbers while others do not If the database used to identify the protein does not have accession numbers this category is not created e Hit Table The annotation entry in this category is the entire list of proteins identified for the spot e Protein Probe This file name category provides a link to the Protein Probe file for the spot e Mass Spec Score Both the Joint Probability and MOWSE scores are listed 10 4 Mass Spectrometry Analysis i Annotation Tool masslynx test ES Select Category y Select Spot r nnotation Entry NN E cr Hit table MALDI 2103 aj ASAJZ2684 NID gZ623983 Escherichia coli 13 peptides 13 92 coverage 115 93kDa score 20617 3 q Protein name MALDI 2202 BETA GALACTOSIDASE EC 3 2 1 23 LACTASE E ESCHERICHIA C m Mass spec score MALDI 2201 allah ast he
421. rom then use the Integrated Excision tool to perform the cuts PDQuest tries to locate the cutter If you are not connected to a cutter you will be prompted to use a simulation Fig 9 1 Fluorescent spot cutter Finally you can export the completed 1 e after cuts are done cut list to Micromass s MassLynx software for mass spectrometry analysis see the next chapter 9 1 PDQuest User Guide 9 1 Excision Gel Selection The Excision Gel Selection tool is used to select the gel s in a MatchSet from which to cut spots using Bio Rad s ProteomeWorks spot cutter and the ProteomeWorks Plus spot cutter systems First you must create an analysis set of spots see section 7 1 Analysis Sets in the MatchSet that you want to cut Then you open the Excision gel Selection tool select that analysis set and identify which gels you want to cut from This creates a cut list of spots and gels Finally you use the Integrated Excision Tool to perform the cuts Note You must open the MatchSet containing the gels whose spots you want to cut and create an analysis set out of those spots before you can open the Excision Gel Selection tool With your MatchSet open select Excision Gel Selection from the Identification menu The Excision Gel Selection tool will open and you will be prompted to select from the list of available analysis sets in the MatchSet 9 2 Integrated Excision Tool LL PDQuest x A Select A
422. rop the left margin of your image Crop Left Margin repositions the image in the Excision Tool window to the left Enter a value in screen pixels in the field to reposition the image Note that you must click Done to close the settings dialog and then acquire a new image for this change to take effect Note that cropping the left margin does not change the raw image only the portion of the displayed image It is important to crop so that you can see the right edge of the gel holding area of the platform in order to maximize the area available for cutting Serial Port In the dialog box select the communications port to the spot cutter by clicking on COM 1 or COM 2 8 1 d Spot Cutter Calibration Before you can begin excising spots you must calibrate the spot cutter This procedure requires a coomassie R 250 stained PVDF membrane Basic Excision Tool Note Before calibration you should focus the cutter camera and align the wash station position You should make sure the spot cutter camera image is properly centered using the Crop Left Margin setting Note Ifyou are using the lens filter for UV light make sure that it is installed BEFORE calibration If you add or remove the lens filter after calibration you must recalibrate through the calibration procedure The first dialog box will prompt you to specify whether you have installed the lens filter or not To use in visible mode only calibrate with the lens filter not installed
423. ropriate checkboxes in the Options dialog box See section B 9 Options Save Clicking on Save will open a separate image window displaying the captured image A Save As dialog box will automatically open displaying the default file name for the image which will include the date time and user if known You can then change the file name and storage directory You can also export your image as a TIFF image for viewing with other applications B 8 Exposure Status The Exposure Status bar shows the progress of your exposure If your exposure time is greater than 1 second the status bar display will give you a graphical representation of the remaining time before exposure is complete If the exposure time is less than 1 second the status bar will not refresh itself for each exposure it will remain at 100 percent B 9 Options Click on the Options button to open the Options dialog box Here you can specify certain settings for your ChemiDoc system B 13 PDQuest User Guide Gel Doc Options Fig B 10 Available options in the ChemiDoc acquisition window Click on OK to implement any changes you make in this box Clicking on Defaults restores the settings to the factory defaults DAC Settings Note The default DAC settings are highly recommended and should be changed with caution Appendix B ChemiDoc These sliders may be used to adjust the minimum and maximum voltage settings of your video capture board The minim
424. rotiter plate in the plate holder As each cut 1s being made 1t will be highlighted in yellow on the screen After each cut has been made the cut circle appears in white on the screen Cuts that have not yet been made are circled in green u Basic Excision Tool Step 1 Acquire Image Light on Gel Light off PVDF Acquire Image CUY on Fluon Time secs a ooo Step 2 Specify Cuts bea Add cut Y Remove cut fe Cuts 0 Excisions 0 Plates 0 Step 3 Set Cutter Options X Spin when cutting Cut longer X Spin when ejecting X Double eject C No spin when lifting JBacked gel Tip motion range ETA Gel defaults PVDF defaults Step 4 Set Wash Options _ Station 1 xj Station 2 x Station 3 xj Station 4 Begin R esume ZE Pause Setup 432 aja Simulate X Cutter x Camera Fig 8 11 Cuts being made 8 17 PDQuest User Guide Confirming Cuts For visible spots inspect the gel to confirm that each cut is being made correctly For fluorescent spots you must take a picture of the gel using UV light to ensure that cuts are being made correctly After one or more cuts have been made click Pause to pause the cut run Note that the cutter will pause only after the most recent cut has been deposited in a well Confirm the pause then click the Confirm Cuts button under the main acquisition window 1 Confirmation Image 1 x1 2D Scan Cut 1 A
425. rrent Master image while outside master shows the number of spots that fall outside that area Check Mark Spots to be added to select the exact position in the master gel you want to add the spot Check Mark add spot location in Master to mark the spot in a member you want to add to the Master Check Auto configure MatchSet subwindows on launch to automatically configure the subwindows in the MatchSet to imitate the Master You can select the style color and symbol for how you want your spots to display by clicking the Format button When you are ready to add spots from the gel click the Proceed button The selected spots will be added and the gel will be rematched with the Master If some spots could not be added because they were too close to existing spots you will be prompted to rematch the gels by a pop up box You can add these spots manually To remove all spots added from the selected gel click Undo Add You will be warned before the action is completed Manually Adding Spots To manually add spots from a member gel to the Master first display the unmatched spots in the gel using Show Symbols F7 or Mark Unmatched Spots Shift F8 Use the Zoom Box tool to magnify a spot 5 28 Matchsets O Select Add Spot to Master from the Match menu or toolbar and click on the center of the unmatched spot in the member The cursor will jump to the same area in the Master calculated from the surrounding matches When you click in
426. rs of the MatchSet except when creating a Master out of gels with different pH gradients see 5 6 Cybergels Avoid spots in clusters in case the spot detection mechanism has misidentified the peaks in the cluster Unless you are creating a Master out of gels with different pH gradients you should landmark spots in all corners of the gels as well as the middle of the gels To begin landmarking first magnify a region of the MatchSet gels and identify a well resolved spot that appears in all the gels Study the region under different levels of magnification to make sure you have identified the same spot in all the gels Matchsets ll Matchset mpdi00187 Modified Al ES This appears as one spot in some gels two in others This spot makes a better landmark because it is _ i H Match symbols matched S wonatch Match symbols matched 3 unmatch Match symbols matched 3 unmate well resolved in STD ampdi00137 std Modified 3679 vO x0 Gasssiar mpdid0187 3686 vO x0 Gaussian mpdidO187 all gels se e t Match symbols matched 3 unmatchH Match symbols matched 3 unmate 3553 v x Gassiarimpdi01S7 35690 vO x Gaussiani mpdhOOls Fig 5 10 Selecting a spot to landmark Select Landmark from the Match menu Match toolbar or right click menu and click on the spot in the MatchSet Master The cursor will jump to the same area in the first member gel If the landmarking function can identify the sa
427. rt aacomp Aming Acid Composition aarat r Amino Acid Patios aasedqys Amino Acid Sequence antibod intibodies to Protein assoprot fAssociated Proteins bindpropr Binding Properties chromloc Chromosome Location Clonvec Cloning Vector coreg Co Regqgulation with Other Prote crystruc Crystallograpic Structure dnaseg DHA Sequence drugsatft Drugs that Affect Protein ecnamber E C Number envaf fecr Environmental Conditions th enzynat Enzymatic Activity pl Cancel 2 Help Fig 2 22 Export Annotations pop up box Click an individual category name to select 1t or shift click or ctrl click to select multiple categories When you have made your selection s click Export In the file name dialog enter a name for the export file and select the directory to save to The data in the text file is tab delimited Each row includes the annotation category SSP number for a spot in that category and the associated annotation entry Text files can be opened in standard Windows and Macintosh documents and spreadsheets If you selected multiple categories to export the annotations will be sorted by category 2 34 General Operation 2 Preferences The Preferences dialog box allows you to customize basic features of your system Select Edit gt Preferences to open the dialog Tabs for Misc Paths Display Toolbars Application and Imagers display 1 Preferences N x Misc Paths Display Toolb
428. s Explorer to open the file 2 3 6 MatchSets Scansets and Analysis Sets MatchSets A MatchSet is PDQuest S mechanism for comparing and analyzing the spots in an experiment Each image in a MatchSet is called a member A MatchSet contains one or more separate members in the form of Raw 2 D Filtered or Gaussian images Using a MatchSet you can make quantitative and qualitative spot comparisons across gels calculate molecular weight isoelectric point values annotate spots group them into analysis sets combine the data from multiple gels into replicate groups and select spots for excision and mass spec analysis PDQuest User Guide A MatchSet can consist of one gel or many gels depending on the type and size of the experiment The MatchSet is displayed in a single window with subwindows for the images of the member gels and a subwindow for the MatchSet Master Ll Matchset MS500004 2001 09 06 Fig 2 6 Example of a MatchSet The commands for creating MatchSets are located under File gt New MatchSet and as Step 3 of the Automated Detection and Matching dialog Scansets The members of a scanset are three separate files of the same root image You can open and close scanset images 1 e different exposures or versions of the same image individually even though they are loaded into the same window 2 8 General Operation sample vl x1 Rav 2 D Image a samplel yl Filtered Fig 2 7 Example of a Scanse
429. s box resize the window make any zoom or transform changes using the icons on the top bar above the window then click on the Recuts icon to bring the recuts box back up E mi Integrated Excision Tool NONE Aman Cut list METAS ET Ioj x Analysis set name Analysis set spot coun ox TP wen S5 Status completed Cutter options X Spin when cutting C Spin when ejecting aoz fe l al gl Fig 9 22 Integrated Excision Tool fluorescent mode re cuts Step 13 Enter the well numbers of the missed pick up spots in the Re cuts box on the screen Step 14 Click on Begin Resume after the selections are made 9 35 PDQuest User Guide 9 2 d Exporting the Cut List You can export the information in the cut list as a spreadsheet file Click the Export button enter a name for the file and select the directory in which to create the file The export command will create a tab delimited file that can be opened as a Microsoft Excel worksheet or as a simple text file 9 36 10 Mass Spectrometry Analysis After you have cut spots from a gel using the Integrated Excision Tool and digested them you can select the parameters for processing them using Micromass s MALDI or MS MS mass spec instruments You can then import the mass spec data back into PDQuest in the form of annotations 10 1 Creating a MassLynx Batch File After you h
430. s eam even nw GR a cla 6 29 6 9 Standard Spot Numbers siii di undmenanend NO ENEN 6 32 6 10 Database Spot Numbers caidas 6 34 Analysis Sets and Annotations ccccssseesseseeeeeenseeeeenes 7 1 Tolo AMANSTS Eo ocic 7 1 Ela AMO LAO TOO AE E cairae laicas 7 22 732 BLOW SINS ADO LAOS di ae 7 33 7 4 Creating Annotations from Analysis Sets occccccccncnnnnnnnnnnnnnnnnnnnnnnnnonononnnos 7 34 POS PARIS ANNONS A ca tactanaviandeno nse Aeneas 7 36 7 6 Transferring Annotations ld 7 37 BaSIC EXCISION TOON cursis 8 1 Sl Spot Cutter Seto sap 8 2 2 CUNE SPOS eso 8 9 PDQuest User Guide 5 Other Spot Cutter Controls ts 8 24 9 Integrated EXCISION Tool oooccconncconcncconcncnenanononancenannnananennas 9 1 DL EXCISTOM GEL ClEC MON a a a 9 2 9 2 Integrated Excision Tola id 9 9 10 Mass Spectrometry Analysis ccccssseesssseeseeeeeesees 10 1 101 Creating a MassLynx Batch Bilal aiii 10 1 10 2 importo Mass Eyn Results anana 10 3 10 3 Mass Spee Score Overlay ia ti 10 5 TOA or o A A a ead ese she GiteawyaaGiaeeaieasse 10 6 1035 Tmport Mascot Results arios race cis 10 7 11 Graphs and Reports cnnnccccccconccccnnnccnonnnccnnannnnannnenaneranannnnas 11 1 isle Conti eure Graphs urnas 11 1 EA MAISTO or atin Grp traida 11 4 EsAB OVN aV aida oder lic clear ccoo 11 7 11 4 Quant Table Repo e cacas 11 9 LS Master Image REDO std ici 11 13 M6 Scale Plot REPO aiaro A T 11 14 Appendix A Gel DOC 2000 00 rica A 1 Ap
431. s folder in the PDQuest folder These sample images are related and can be used to create a MatchSet Opening TIFF Images The Open command can also be used to import TIFF images from other software applications TIFF images do not contain all of the tagged information that would normally be included in a Discovery Series image file e g scanner type date color etc For this reason the Image Info and Print gt Image Report commands will not display all such tagged information from TIFF files There are many types of TIFF formats that exist on the market Not all are supported by the Discovery Series There are two broad categories of TIFF files that are supported 1 8 bit Grayscale Most scanners have an option between line art full color and grayscale formats Select grayscale for use with the Discovery Series software In a grayscale format each pixel is assigned a value from 0 to 255 with each value corresponding to a particular shade of gray Hewlett Packard Microtek and Sharp each make scanners that produce compatible 8 bit grayscale images 2 12 Note General Operation 16 bit Grayscale Bio Rad s Molecular Imager storage phosphor systems use 16 bit pixel values to describe intensity of scale Molecular Dynamics imagers also use 16 bit pixel values The Discovery Series understands these formats and can interpret images from both Bio Rad and Molecular Dynamics storage phosphor systems PDQues
432. s of each exposure Depending on which dark subtraction type you have selected under Options a dark image may be acquired immediately following each exposure When each exposure is complete an image window containing that exposure will open in front of the VersaDoc window Subsequent exposures are tiled in front of the VersaDoc window Note that the first exposure will have the base file name the default base file name is the computer user name and a time stamp Each subsequent exposure will have a version number v2 v3 v4 etc appended to the base file name If you are using the K 13 PDQuest User Guide default base file name the time stamp may change in the course of the series of exposures in this case the base file name will change and the version numbering will reset for subsequent exposures The highest version number will be the final exposure If you did not elect to auto save the exposures as they were created then each image will be unsaved To stop the image acquisitions click on the Stop button Note Exposures captured before stopping will be displayed in image windows Study the different images and select the best exposure s to keep K 6 Options Click on the Options button to open the Options dialog box ersaDoc Options i x Information Dark Subtraction Type f Normal 7 Referenced None Reset Referenced Mode Versaboc 5000 Firmware Version 1 0 Hardwa
433. s only affect image windows not subwindows Select Tile to resize all open windows so that all the images are maximized The windows will be arranged on the screen from left to right and top to bottom Tile Vertical will resize all open windows and arrange them in columns across the screen Tile Horizontal will resize all open windows and arrange them in rows across the screen 3 5 Magnifying Images The magnifying tools in PDQuest can be accessed from the main toolbar or View menu or by key command Note that by default if you magnify a region in one image of a MatchSet or scanset the same region in all the other subwindows will be magnified To turn off this feature deselect the Auto All Same checkbox under Display Preferences Edit gt Preferences Ctrl F3 will undo your last magnification or other viewing command 3 4 Toolbars view Entire Image oom Box Grab oom In oom UE View Density Hide Overlays Advanced view Multi channel viewer Viewing and Editing Images ab ER SSS 080 M Fl Fe Cho Alk F2 Ctrl F2 F4 Fig 3 3 Viewing functions on View menu and main toolbar Zoom Box Use the Zoom Box tool to magnify a specific region of an image MatchSet or scanset Select Zoom Box from the main toolbar or View menu or position your cursor on the image and press F2 Drag a box on the image then release the mouse button The area you selected will be magnified 3 5
434. s size J 4vg norm gly x Norm coeff of var Member gel count Fig 2 20 Spot data by group tab Basic Export MatchSet The Basic Export MatchSet dialog allows you to export the individual characteristics of spots in a MatchSet This is a more basic version of the standard Export MatchSet dialog Data will be exported in ASCII text format With the MatchSet loaded select Basic Export MatchSet from the File gt Export submenu 2 31 PDQuest User Guide Basic Export Matchset Dete lia os coord s sigma peak value spot quantity export Cy coord y sigma spot quality Spots ta All spots a Completely Spots in export matched spots Analysis Set Spot Count 570 Saturated A 7 S 3 E stimate 3 0 b Specify 3 0 Unmatched EE 22 Minimum Spot 1 0 b Specify Er Field Separatorz Commas 1 2 3 format f Tabs Excel format Export Destination fe File 7 Clipboard yf OK X Cancel 2 Help Fig 2 21 Export MatchSet Data dialog box Next to the Data to export options select the characteristic of the spot that you want to export Specify the spots whose data should be exported You can choose either all the spots in the MatchSet those spots that are completely matched in every member of the MatchSet or the spots in a particular Analysis Set The Spot Count automatically adjusts to reflect the total number of spots selected If you are exporting spot quantity data select either es
435. s staff You can also use Solobug installed with PDQuest to make software feature requests xii 1 Introduction 1 1 Overview of PDQuest PDQuest is a software package for imaging analyzing and databasing Raw 2 D electrophoresis gels The software runs in a Windows or Macintosh environment and has a graphical interface with standard pull down menus toolbars and keyboard commands PDQuest can acquire images of gels using any of several Bio Rad imaging systems An image of a gel is captured using the controls in the imaging device window and displayed on your computer screen The scanned gel can then be cropped rotated etc using the image editing controls Spot Detection Analysis and Databasing With Automated Spot Detection and Matching you can select the gels you want to anlayze detect spots of interest create a MatchSet and match gels all from one dialog box The Spot Detection Wizard guides you through the process of identifying and quantifying the spots in the gel image After detection gels in the same experimental series are placed in a MatchSet for comparison statistical analysis and databasing Histograms allow you to quickly compare the quantities of the same spot in all the gels in a MatchSet Spots can also be compared qualitatively organized into user defined sets for further analysis and annotated and databased for easy identification Spots from different experimental series can be organized and compared in
436. s you added For more information on selection criteria formatting spot location and display 5 7 Adding Spots to the Master Indicate the orientation in which your gels are run by checking either the Acid Base or Base Acid box under pH gradient in the Edit gt Preferences gt Application tab When you are ready to add spots from the gel click Proceed The selected spots will be added and the gel will be rematched with the Master If some spots could not be added because they were too close to existing spots this will be noted in a pop up box You can add these manually To remove all spots added from the selected gel click Undo Add You will be warned before the action is completed 9 25 PDQuest User Guide Landmarking Gels with Different pH Gradients If you are combining gels with different pH gradients into the same Master you will not be able to landmark the same spot in all the gels For example to create a cybergel from three gels with different pH gradients you would place landmarks in the overlapping region of the first two gels then add the spots from the unique region of the second gel to the Master Then you would landmark the overlapping region of the last two gels 5 Adding Spots to the Master Recall that all spots initially included in the MatchSet Master come from the template gel used to create the Master After you have matched these spots in all the gels you are ready to add the unique spots from the other
437. sBase Options dialog a program and or project will also be required If the images in step one are located in a project and or an experiment these fields are automatically filled in with the project and or experiment where the images are located To select a different program project or experiment or if the images are not located in a program project or experiment click the field label to open the Open from WorksBase dialog Locate the appropriate program project or experiment and click Select Note If you want to detect spots without creating a MatchSet check the Disable this step checkbox at the bottom of Step 3 Step 4 Match Gels This is similar to Step 2 in that you may select existing parameters for matching or you may create new ones with the Edit button However unlike Spot Detection parameters the matching parameters work over a wide range of images and you will probably obtain satisfactory results with the default parameters Note If you want to detect spots without matching gels check the Disable this step checkbox at the bottom of Step 4 PDQuest User Guide When you press Go all the images will be loaded from WorksBase and processed Once the spots have been detected a MatchSet will be created in WorksBase and the gels will be automatically matched Note You cannot use Automated Detection and Matching in WorksBase on gels containing previously excised spots You will receive a warning and processin
438. sample for the run you may press View identifications to launch WorksBase for Proteomics protein viewer for this sample Note that WorksBase for Proteomics must be running for this to work Applying results You may select Apply all to automatically import the hit with the highest score for each sample Alternatively you must select a hit from the hit list and press Apply This may be used if the hit with the highest score does not appear to be correct N 8 Example identifying spots from a single gel A fairly typical special case is where a user wants to simply identify some spots from a single gel The analysis tools in PDQuest are not needed the user knows which spots to identify from some other means Starting with the gel Step 1 Step 2 Step 3 Step 4 Acquire an image of the gel Save the image in WorksBase Run Spots Automated Detection and Matching Specify the single gel N 22 Step 5 Step 6 Step 7 Step 8 Step 9 Step 10 Step 11 Step 12 Step 13 Step 14 Step 15 Step 16 Step 17 Step 18 Note Appendix N WorksBase Specify the detection parameters and MatchSet name Perform automated detection and matching the MatchSet opens showing the master and the single image Right click on the master select Analysis Set Manager Click Create Select Arbitrary from the Select Set Type dialog Enter a name for the set Select No spots as the
439. select it from the application tree by selecting Custom and the name you created You can delete the application by selecting Custom Delete and the name of the application Radioisotopes Chemiluminescence Chemifluorescence DNA Stain Gel Protein Stain Gel Fluorophores Microtiter Plate Densitometry 4 Custom Create Delete Custom Custom Application 1 Medium Sample Intensity Step Il Select Scan Area Low Sample Intensity Click and drag in diagram to set scan area High Sample Intensity Fig J 7 Selecting a custom application You can edit a custom application by selecting Custom Edit and the name of the application You can also use this feature to create a new custom application from an existing one Step Select Application Y Channel 1 Channel 2 Channel 3 Channel 4 Select Densitometry Coomassie Blue Gel s Blot Medium 532 1l06dnm EX Blank 555nm LP 1l0 4nm BL Fig J 8 Application selection and settings J 10 Appendix J FX Once you select an application the application name and settings appear below the Select button J 3 Step Il Select Scan Area To select a scan area drag your mouse within the scanning window In the scanning window your cursor appearance will change to a cross The border of the scan area you are selecting is marked by a frame Fig J 9 Selecting a scan area If you are in quadrant mode note that the frame locks
440. significantly over the past several years with the advent of IPG strips and simplified gel running techniques large format and cyber gels that allow for greater pH range and specificity and new stains and staining techniques In addition mass spectrometry now allows for peptide mass fingerprinting of very small amounts of protein isolated from gels Using a combination of these techniques pharmaceutical companies can now use Raw 2 D technology for high throughput screening of drug compound candidates using protein targets research laboratories can study large scale changes in protein Preface expression and companies and institutions can cross identify and catalog hundreds of thousands of protein species at the cellular level This provides an excellent technique for the study of differential gene expression under various growth conditions Since the expression and regulation of individual proteins can be detected Raw 2 D gels are an indirect way to monitor gene activity They allow for the investigation of quantitative as well as qualitative changes in cellular protein expression The environmental conditions of a cell can be changed in order to determine optimal growth conditions as well as monitor the cell s response to different stresses Environmental conditions that can stress the cell include changes in temperature pH and nutrient availability Some examples of the chemical stresses that can be placed on a cell include drug and horm
441. sis Tools analysis sets based on the attribute values See Section 7 1 Analysis Sets for information on creating analysis sets mage Sample treated stock samplel Yes 30 Deg Red stock samples Ho Lie Yes 20 Deg Yes hai Sample6 No 40 Deg Rea Fig 6 17 Sample Report showing attributes and values Yes No 20 Deg Yes Yes All of the samples in the above table have the attributes treated temp and color The samples with the attribute value yes in treated are considered members of a class The same goes for the samples with the attribute Temp and the same value A gel can belong to as many classes as it has attibutes Once you have associated samples to gels and added attributes to them you can use the classes when creating analysis sets See Section 7 1 Analysis Sets for information on creating analysis sets 6 6 c Copying a Sample Database from another MatchSet To copy the sample database of another MatchSet into the current MatchSet select Analyze gt Describe Samples gt Copy Sample Database If there is data already in your database PDQuest confirms your request to overwrite the current database Click OK This opens the Select Files to Import dialog Locate the MatchSet containing the database you wish to import and click Open 6 6 d Sample Pairs Sample Pairs are used for statistical analysis of sample proteins To create sample pairs select Analyze gt Describe Samples gt Pair Sampl
442. sitivity or visa versa there will be a delay of several minutes while the Fluor S MAX camera cools down or warms up If you attempt to acquire an image during this period you will be notified of the changing temperature If you do not want to wait you can cancel the mode change H 6 Appendix H Fluor S MAX H 3 Step Il Position Focus Note When you click on the Position or Focus button the light inside the Fluor S MAX box automatically turns off This is because if you focus with the camera at maximum aperture see note below leaving the light on would make it difficult to view the image To turn the light on while positioning or focusing hold down the SHIFT key when clicking on the button Position The next step in acquiring an image is centering your gel or other object within the camera frame To do this click on the Position button The Fluor S MAX will begin capturing a live image and updating it every second With the Position button selected look at the image in the acquisition window while you position your object in the center of the platen If you have a zoom lens on the camera you can adjust the magnification while you position You can select the Show Alignment Grid checkbox to facilitate positioning Fig H 7 Fluor S MAX alignment grid When you are finished positioning click on the Stop button Focus Note Before focusing you should adjust the f stop on the camera to the lowest setting 1 e t
443. spot This takes much longer and will cause MatchSet saves to take a considerable amount of time Again this is only required if you want to read spot information with other programs e Save all spot details e Save matched spot details e Save annotated amp excised spot details Recommended e Save excised spot details The Save gaussian raster image checkbox causes a normal pixel raster image to be saved in the database This image is then available to other programs that want to display the image without having to understand how to generate pixel raster image from the list of gaussian models Generating this file is not required by PDQuest N 5 PDQuest User Guide Login The Login tab of the WorksBase Options dialog allows you to specify the default user name and password to display in the login dialog when you connect to WorksBase li WorksBase Options E x Connection Save Login Display WE Username prradison WE Password rare Database E Purpose Read only e Head wnite Administrator Status wf Done X Cancel Z Help Fig N 4 WorksBase Options Login tab The Purpose section is not operational at this point Display The Display tab of the WorksBase Options dialog allows you to customize the way WorksBase looks and functions in PDQuest N 6 Appendix N WorksBase Ll WorksBase Options Eoo x Connection Save Login Display Explorer x Sho
444. st free well aot Specify starting well x Station 3 G Current run serial f OK X Cancel Select microtiter plate cay ES Confirm vf Done x Cancel elect Microtiter Plate 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 WE 1 1 Cutter options xX Spin when cutting _ Cut longer X Spin when ejecting X Double eject _ No spin when lifting JBacked gel Tip motion range E o Gel defaults PYDF defaults Align Ea A Camera sell E ExT Close a Help Fig 9 4 Select Microtiter Plate pop ups The Select Microtiter Plate pop up prompts you to specify the plate number and size for the current cut run In the dialog box a default plate name will appear in the Plate Name field Note that default plates are named sequentially Plate 001 Plate 002 etc However you can enter any name you want in the field Then specify the well you want to use as the First free well using the Specify starting well button This button opens the Enter Plate Information pop up box If you specify a starting well other than the default of Al the skipped wells will be noted in the box Click Done to make your selection and OK to complete the plate selection process Note Specifying a different starting well is essential if you want to load spots from different MatchSets or different analysis sets on the same microtiter plate You 9 10 Integrated Excision
445. symbol color is green Default Match Settings Matching settings De fault Match Settings Cancel Members all Matches wea Extensions ura Sum ura 9 Select Member to be matched samples vl Gaussian 161 spots i Automated Matching Matchset openheart sample2 v2 Gaussian 145 spots sample3 vl Gaussian 466 spots Primary Matching Extended Matching sampled vl Gaussian 650 spots Choose the method which will be used to extend the primary matches FEB FI AS 194 spons samplel vl Gaussian 281 spots Extension method Disabled vY Cancel strict liberal Matching Stringency i G Match symbol color is blue i PDQUEST xi Matching settings De fault Match Settings A Select Method Members a11 Disabled Matches ura Extensions wea Sum za Restricted Classic Match A Match Y Done X Cancel C Help Fig 5 12 Automated Matching Primary and Extended options Use the Primary Matching tab to find landmarks automatically and calculate an accurate matching The color of the match symbol for primary matching is green Check the Enable overlapped pH range matching box to create a cybergel 5 20 Matchsets Set the matching precision slider towards strict or liberal depending on the degree of precision you prefer The most rigid setting you can choose is O and the most flexible setting you can choose is 100 The number 50 is the
446. t A scanset consists of three separate images of the same gel image They are displayed in subwindows as a Raw 2 D Image as a Filtered image and as a Gaussian image All of these are saved as separate images but they are part of the same scanset They have the same root file name and when you open them they are loaded into subwindows of the same image window To perform an operation on a particular image in a MatchSet or scanset simply move your cursor over its subwindow A green border appears around the selected subwindow The border shifts as you move the cursor over different subwindows Alternatively you can select Click to Focus Subwindow under Edit gt Preferences gt Display in which case you must click on a subwindow to select it For example the following image files are all in the same scanset Proteins v1 x2 gsc Raw 2 D scan exposure 2 Proteins v1 gim Filtered image Proteins vl gsp Gaussian image PDQuest User Guide Analysis Set An Analysis set is a set of spots you have chosen to study Analysis sets allow you to create groups of spots that are statistically and biologically significant For more information see Chapter 7 Analysis Sets and Annotations lid Matchset MSO0002 2001 10 04 Modified Analysis set ahmed 5 a _ A PTY MS00002 2001 10 04 Master sampleS vl xl Raw 2 D Image gt gt sample4 vl xl Raw 2 D Image Fig 2 8 Analysis Set 2 3 C Opening Files
447. t select De Activate Segment again and click on the segment The segment will be reactivated and included in calibration curve calculations L 1 e Repeat for Each Calstrip If you have multiple film exposures of the same gel repeat the steps outlined above for each exposure The calibration strip batch number for each film exposure of a L 9 PDQuest User Guide single gel should be the same since the same physical calibration strip was used to make each exposure L 2 Merging Multiple Exposures If you are working with 8 bit images and are using calstrips on your scans you can merge multiple exposures of the same gel into a single image with a greater dynamic range Note Youcan only merge 8 bit images with calstrips If you are not working with 8 bit images or are not using calstrips you cannot merge exposures To merge multiple exposures make sure all your exposures and calstrips are loaded into the same image window then simply auto detect spots as you would normally see the following chapter Creating Different Exposure Files To identify different gel scans as different exposures of the same gel open the scan files select Change Version Exposure from the File menu and enter consecutive exposure numbers for the different scans Make sure that the root names of the scan files are the same Note You can give different exposures the same root name when you are scanning them or you can change them later using the Save As
448. t User Guide ab a M ate hes a E n saa eua case anes E 5 32 POP UPS 11 6 EA y PC nn E E 11 7 A o A 11 4 Display order of histogram bars ooooooococonocooonononononanononanonnnnnnnnnncnnnnnnnnccnnnnnnnnos 11 5 Group CONCA US esan hanced 6 13 GS 700 Imaging Densitometer ApPphcatons AA O T aad D 4 CAMDEN ais D 10 Calibratto Setmes ati otra D 14 Filters and heht source SCICCUIMG mins leidas D 5 O D 14 CON CES ALIN LIN a loba D 14 PRE VIC WSC D 6 Resolution SE IEC UMC cerrara rated iia E D 7 Saturated pixels highlighting scan a os D 15 Scan aro selecto illo iuris D 7 is AA a a A a a D 9 SCANNE WING OW otrora ollas D 3 SD CO Car lis D 1 STMT AON MOE dis lso D 1 SUC Pla DICE EdE a ta co D 11 GS 710 Imaging Densitometer Applications SEIECHAS creais E 4 COND RAO yoe S E N E 9 Calibration SLUGS art E 13 Filters and le ht Source seleccin mini ile iio E 6 OPONSE E saiaasecnans maar E ces nean one E 14 CON CTS ALIA IN a a dao oios E 14 PRE VICW SC I a EE E E E 7 Resolution selec NE aaan r a o E ei E 8 Saturated pixels highlighting sn lo E 15 Scan aro selecto libro outs E 7 SCANNE AA lod E 13 PPP A E 3 SES ranita E 1 STMT IA On MOE dois lies E 1 UE Pita DICK dni illa E 10 GS 800 Imaging Densitometer Index 8 Applications selectins ls acid dica di F 4 CAND EAU ON dns aman ele ia F 9 CaM DratlOm Se TINGS sii d F 13 Calibration step Tables iras oe cit F 10 Filters and light source selecting oooooooonnnnnonoonnonon o
449. t can import 8 and 16 bit TIFF images from both Macintosh and PC platforms PDQuest cannot import compressed images TIFF files that are not supported include l 1 bit Line Art This format is generally used for scanning text for optical character recognition or line drawings Each pixel in an image is read as either black or white Because the software needs to read continuous gradations to perform gel analysis this on off pixel format is not used 24 bit Full Color or 256 Indexed Color These formats are frequently used for retouching photographs and are currently unsupported in the Discovery Series although most scanners that are capable of producing 24 bit and indexed color images will be able to produce grayscale scans as well Compressed Files The software does not read compressed TIFF images Since most programs offer compression as a selectable option files intended for compatibility with the Discovery Series should be formatted with the compression option turned off Export Image as TIFF The Reports gt Export to TIFF dialog box contains the settings for exporting a Raw 2 D scan Filtered image or Gaussian image in TIFF format 2 13 PDQuest User Guide ul Export samplel Export Mode Analysis esport raw data Publishing e export view excluding overlays export view including overlays Resolution dpi f 72 dp Same as scan 145 1 150 dpi 7 Specify 72 o 300 dpi
450. t image Click on the Live Acquire button A settings dialog box will open in which you can specify the total exposure time starting exposure time and number of exposures B 8 Appendix B ChemiDoc Chemi Doc Live Acquire Live Acquire Settings Total Exposure Time sec 1o 00 Starting Exposure Time sec ls 00 Number of Exposures ls Save Images Cancel OF Fig B 7 Live Acquire settings Note You should specify no more than 10 exposures in the Live Acquire Settings dialog to avoid excessive build up of image background in later exposures The fewer the exposures the less background will be added to the image See the Release Notes for additional instructions on reducing background in images captured using Live Acquire Select the Save Images checkbox if you want to automatically save each exposure as it is taken Click on OK in the settings dialog to begin taking exposures If you selected Save Images a Save dialog box will open in which you can specify the base file name and location of the exposure files When you click on Save the exposures will be taken The specified number of exposures will be taken at equal intervals between the starting exposure time and total exposure time When each exposure is complete an image window containing that exposure will open behind the ChemiDoc window When the full exposure time has lapsed all the image windows will move in front of the ChemiDoc window Note tha
451. t marks 2 Cut area we Be Ll i i Matches Multiple cuts Light on Gel H el B Jump and Contimm Confidence at Spot 72 ey Li a Light off PVDF Confidence Threshold A E xJAuto zoom x Data box Compute cut overlaps Cut overlaps UY on Fluor Time secs Step 3 Perform Alignment Cuts below confidence 0 Total cuts 2 Bra Manual Correction drag any cut mark on camera image anytime Q Begin Resume Cutting SE Cancel 4 Help Fig 9 9 Aligning the images In the Align Images dialog the left window displays the MatchSet image and the right window will contain the cutter camera image of the corresponding gel 9 23 PDQuest User Guide Note You always have the option of manually correcting spot positions Auto alignment is not always correct and the manual collection allows you to readjust your spot positions Flag the spot you want to correct in the cutter camera image and drag the spot to the correct position Red indicates the spot is below the confidence threshold green spots indicate the spot is above the confidence threshold and the spot becomes white while it is dragged You also have the option of returning to Data Box Positioning Step 1 Light Options To acquire this image first select the light option appropriate for your gel membrane stain type e Light on gels e Light off PVDF membranes Ambient light will produce a better image for PVDF membranes
452. t the first exposure will have the base file name the default base file name is the computer user name and a time stamp Each subsequent exposure will have a version number v2 v3 v4 etc appended to the base file name The highest version number will be the final exposure If you did not elect to auto save the exposures as they were created then each image will be unsaved B 9 PDQuest User Guide To stop the Live Acquire click on the Freeze button or adjust any of the subsequent controls e g Video Print Image Mode Display controls etc Note Exposures captured before freezing will be displayed in image windows Study the different images and select the best exposure s to keep You can then proceed to the next step B 5 Step IV Optimize Display The Display controls are useful for quickly adjusting the appearance of your image for output to a video printer Adjusting these controls will automatically freeze the video display and allow you to alter the image within the ChemiDoc window Step V Optimize Display High 2zss Low lo Gamma 1 00 Highlight Saturated Pixels lnvert Display Auto scale Reset Fig B 8 Display controls These controls are similar to those in the Transform dialog box Note The Display controls will only change the appearance of the image They will not change the underlying data High Low Sliders If Auto scale doesn t give you the appearance you w
453. table in blue The table will initially show one cut request per spot that is one highlighted blue table cell per row If you click the Standard Spot Number SSP at the beginning of each row that spot will appear highlighted in the actual MatchSet See Figure 9 2 Excision Gel Selection for a pictoral representation Study the size and quality of each spot in the gels in the MatchSet when selecting spots to cut You can manually add or remove cut requests by clicking on the table cells Click on a blue cell to delete a cut request Click on a white cell to make a cut request To cut the same spot in different gels click on multiple white cells in the same row they will Integrated Excision Tool be highlighted in blue You can add as many cut requests in as many gels in the MatchSet as you want At the bottom of the table the total number of cut requests per gel is shown 9 1 d Performing the Cuts When you have made your selections click the Next button to close the Excision Gel Selection tool and open the Integrated Excision Tool 9 2 Integrated Excision Tool The Integrated Excision Tool is used to cut a set of spots from the gels in a MatchSet using Bio Rad s ProteomeWorks spot cutter and ProteomeWorks Plus spot cutter systems It provides all the features of the Basic Excision Tool plus the ability to cut spots from PDQuest MatchSets Before using this tool you must have selected the spots and the gels to cut using
454. tation of the remaining time before exposure is complete If the exposure time is less than 1 second the status bar will not refresh itself for each exposure it will remain at 100 percent A 9 Options Click on the Options button to open the Options dialog box Here you can specify certain settings for your Gel Doc system PDQuest User Guide Gel Doc Options Mar 130 gt Imaging rea cm width 20 00 Height 15 00 Auto Exposure Threshold Z Saturated Pisels o 15 Reminder x Show Light Warning Video Printing Footer Information x File Hame x Date Time x Exposure Time X Contrast Settings Save Options Make Backup Copy Misc Options _ Simple Acquisition Mode Defaults Cancel OF Fig A 9 Available options in the Gel Doc acquisition window Click on OK to implement any changes you make in this box Clicking on Defaults restores the settings to the factory defaults DAC Settings Note The default DAC settings are highly recommended and should be changed with caution These sliders may be used to adjust the minimum and maximum voltage settings of your video capture board The minimum slider defines the pixel value that will appear A 12 Appendix A Gel Doc as white in the image while the maximum slider defines the pixel value that will appear as black The slider scale is 0 255 with the defaults set to 60 minimum and 130 maximum Imaging Area Thes
455. te and apply multiple analysis sets With the ASM you can create and apply one or more analysis sets at a time edit one analysis set at a time quickly switch analysis sets on and off and delete an analysis set The ASM also allows you to determine when to apply the analysis sets which gels to mark and what overlays to include in your analysis To open the ASM select Analyze gt Analysis Set Manager 7 2 Analysis Sets and Annotations iul Analysis Set Manager M500002 2002 07 02 Mark Syml Analysis set name on a x X Quali gt E auant 2 x arbita Show x window labels _ 55F number Auho eyvaluate On opening MatchSet gt Alter any edit Mark spots on Master gel f Operand gels f All gels yf Done Gee waluate Al Help Fig 7 1 Analysis Set Manager Format The top portion of the analysis set lists the analysis sets associated with the current MatchSet Mark A check in this box indicates the analysis set is currently applied to the MatchSet To hide an analysis set uncheck the box Click the column header to organize the list according to marked sets Sym indicates the spot symbol for the analysis set on the gels you specify Click the symbol to open the Spot Symbol Format dialog From this dialog you can choose style color and symbol Analysis Set Name Indicates the name of the analysis set Click the column header to organize the list according to
456. te click on the Auto Save After Scan checkbox Note In PDQuest this option is preselected and cannot be turned off All images must be automatically saved when acquired With this checkbox selected when you click on Acquire or Live Acquire a Save As dialog box will open asking you to specify a file name and location for the image you are about to create The scan will begin when you click on the Save button Note that in Live Acquire mode you can save your intermediate exposures by selecting Auto Save After Scan and then Save All Intermediate Images Make Backup Copy You can automatically create a backup copy of any scan you create To do so first select Auto Save After Scan see above then select the Make Backup Copy checkbox With this checkbox selected when you save a scan a backup copy will be placed in the same directory as the scanned image Windows backup files will have an sbk extension Macintosh backup files will have the word backup after the file name Note To open a Windows backup file you must first change the file extension To locate the file after changing the extension select All files in the Files of type drop down list of the Open dialog G 6 d Imaging Area Size The imaging area is the area of the sample that is captured by the camera and displayed in the scan window To specify the size of this area enter a dimension in the appropriate field under Imaging Area PDQuest User Guide Whe
457. te filter S20LP 530DF60 610LP clear or none light type UV white or none and light source Trans Epi or neither in the VersaDoc for that particular application Appendix K VersaDoc Nucleic Acid Gels Protein Gels Coomassie Blue Blotting Copper Stain VPRO ange SYPRO Red SYPRO Ruby Zinc Stain Densitometry Custom Serra a Photographs Clear White EPI O 5x Gain 1x1 Bin Fig K 3 The application tree in the VersaDoc acquisition window Note If you select an application that requires trans white illumination you will need to place the white light conversion screen on the sample stage area To exit the tree without selecting press the Esc key or click outside the dialog box Your selection will be displayed below the Select button Custom Applications If your application is not listed if you want to use a user installed filter or if you want to set the gain and bin settings manually you can create and save your own custom applications From the application tree select Custom then Create This will open a dialog box in which you can name your application and select your settings K 5 PDQuest User Guide Nucleic Acid Gels Protein Gels Blotting Channel 3 Channel 4 K Edit Delete Densitometry Custom Colorimetric Clear White EPI f Custom Application 1 lx Gain lxl Bin Custom Application 2 Create anew ers
458. ted image by selecting 72 dpi typical computer screen resolution 150 dpi or 300 dpi standard printing resolutions Same as scan or any resolution you Specify up to the resolution of the scan If you have log transformed the image you can specify a Linear transform or preserve the Current view If your image has a pixel depth of 16 bits you can compress it to 8 bit data for export to TIFF by selecting the 8 bit option Note TIFF images are exported from the Multi channel Viewer in 24 bit RGB mode to preserve the colors displayed in the viewer Finally if you are only displaying part of the image due to magnification or repositioning you can preserve the Current view or export the Entire image Exporting the Image The size of the pixels in the image and the file size of the image are listed at the bottom of the dialog When you are ready to export click the Export button The Export TIFF Image dialog box opens The default file name will have a tif extension and the file type will indicate that this is a TIFF image You can change the file name or select a different directory to save in Click Save to complete the export A confirmation appears in the status box of the top menu bar 2 27 PDQuest User Guide k Export to TIFF Image ahmed tif has been saved on disk Fig 2 18 Status box confirms your export to TIFF is successful 2 6 b MatchSet Data Data from a MatchSet can be exported as an ASCII text file f
459. th the suffix appropriate to the image file type e g 2 D scan For 2 D scans use the suffix gsc For Filtered images use the suffix gim For Gaussian images use the suffix gsp For calstrip scans use the suffix csc For MatchSets use the suffix ms For high level MatchSets use the suffix hms M 2 PC to Macintosh PC versions of Discovery Series files can be read directly by Macintosh applications with no required modifications PDQuest User Guide M 2 Appendix N WorksBase Appendix N WorksBase z The WorksBase options in the File menu provide PDQuest with a database Ir 1 to store organize and analyze scientific data You can also integrate the BL information you generate in PDQuest with other reference databases using WorksBase WorksBase focuses on protein samples 2D electrophoresis and Mass Spectrometry Mass Spec Select Edit gt Preferences gt Enable WorksBase commands to display WorksBase options in the File and the Match menus For more information on WorksBase see the WorksBase manual File Edit View Image Spots Match Analyze Identify Report Open From Disk Save on Disk Save As on Disk Save All on Disk WorksBase Open From WorksBase Save in WorksBase Save Allin orksBase Rename Relink in Wworksbase Save in Place Save ll in Place Revert bo Saved Report wizard Close Alt F4 Close All Login Unload Ctrl U Logout Gel Mnr at F
460. than 10 saturated pixels you will receive a warning message If this happens you can go back and select a higher sample intensity in the application tree Saving the Image After the scan is complete a message will appear asking you if you want to keep the scan If you select Yes a separate window will pop up containing the new image You can then save and analyze the image using the standard menu and toolbar functions J 13 PDQuest User Guide J 6 Options Auto Save After Scan To automatically save any scan you create click on the Auto Save After Scan checkbox With this checkbox selected when you click on Acquire a Save As dialog box will open asking you to specify a file name and location for the image you are about to create The scan will begin when you click on the Save button If you are scanning using multiple channels the image created using Channel 1 will be saved using the base file name and images created using subsequent channels will have the base file name plus a version number v 2 v 3 v 4 Note that the image version number does not necessarily correspond to the channel number For example if you scanned an image using only Channels 1 and 4 the image created using Channel 4 will still be saved as version 2 v 2 Make Backup Copy You can automatically create a backup copy of any scan you create To do so first select Auto Save After Scan see above then select the Make Backup Copy checkbox
461. the Excision Gel Selection tool as described above The MatchSet containing the gels must be open and active Also make sure the spot cutter is connected with the computer is switched on and has warmed up and make sure the camera is connected and has been turned on Note See the spot cutter hardware manual for instructions on installing the hardware and software Select Integrated Excision Tool from the Identification menu The software will connect with the spot cutter instrument and camera and the Integrated Excision Tool dialog box will open 9 9 PDQuest User Guide i Integrated Excision Tool N ES Cut list Member image Analysis set name Demo Qual Gel spots to cut 1 0 Analysis set spot count 1 2 Tatal ante fined reiki a n 1 Enter Plate Information Cut MTP SSP Gel Quantity Run Cuts Cut i d TE O 1 401 4202 Demo 30degreel 3204 5 05 1 eee te Aaa O 1 402 1303 Demo 30degreel 35095 05 L Starting well aor 1 403 8201 Demo 30degree2 25671 l E gt Wells skipped to x 1 404 5401 Demo 30degree2 4185 7 x 1 405 9701 Demo 30deqree2 3490 9 x 1 406 0602 Demo 30degree2 13572 7 x 1 407 0703 Demo 30degree3 2068 6 x 1 408 4401 Demo 30degree3 2491 9 x 1 409 3704 Demo 30degree3 2560 3 x 1 410 7903 Demo 30degree3 2649 3 Select plate to edit 1 y Add new plate Status ERs cna arora ees Plate name PlateOOl Plate size a6 gt Wash options Station Fir
462. the Master the spot will appear and a match will automatically be made Rs Mark Added Spots M Click Mark Added Spots ALT F8 on the menu or toolbar to highlight the al spots you have added to the Master remove a spot you have added manually The cursor displays as a red X which you position over the unwanted spot 5 8 Edit Match Submenu After you have completed adding spots to your master gel the Match gt Edit Matches submenu contains further commands for editing MatchSets Some of these commands can also be located in the Match Toolbar View gt Toolbar gt Match Tools or through the Quick Reference Guide 5 29 PDQuest User Guide Match Analysis Identification Reports Window Help New MatchSet MatchSet Tool Automated Matching Landmark Unlandmark Classic Match Gel Classic Match All Gels Edit Matches Show Symbols FY Show Offsets Shift F Mark Landmarks Fa Show Match Fa Combine Overlapping Gels CyberGel Manual Match Manual Unmatch Mark Unmatched Spots ShiFt F8 Cancel Unmatched Spots Restore Unmatched Spots Mark Partial Matches Graph Partial Matches 4uto 4dd Spots to Master Add Spot to Master Mark Added Spots Alt F6 Remove Added Spot Mark Erratic Responses Graph Erratic Responses Colony two point Match Remove Auto Matches Remove All Matches Fig 5 17 Edit matches submenu commands Manual Matching After you have landmarked numerous spots and review
463. the analysis set name An up arrow in the column header indicates ascending order while a down arrow indicates descending order PDQuest User Guide Count Indicates the number of spots that match the analysis set criteria Click the column header to organize the list according to the Count An up arrow in the column header indicates ascending order while a down arrow indicates descending order Edit Click the button under the Edit heading to either edit the analysis set or delete it from the list The bottom portion of the ASM allows you to determine when to evaluate the MatchSet on which gels to mark spots and what overlays to show Analysis sets are automatically evaluated when you open the MatchSet when you open the Analysis Set Manager and if you make any edits to an analysis set If you have On opening MatchSet selected as your Auto evaluate option click Evaluate after any spot editing Click Done to close the ASM 7 1 6 Creating an Analysis Set To create an analysis set click Create in the ASM This opens a dialog for you to select the type of set you wish to create 7 4 Analysis Sets and Annotations hl Select Set Type x A B Analyses Gl ualitative Gl uantitative Statistic Uther Analyses Arbitrary Boolean Matching X Cancel Z Help Fig 7 2 Select the Analysis Set type you wish to create In the Select Set Type dialog select the type you want to create by clicking the n
464. the light source is uncalibrated reflective and 1 25 top to bottom and 1 20 left to right if the light source is uncalibrated transmissive see below for details To hide the gridlines click on the Hide Grid checkbox under Options PDQuest User Guide The control panel has been arranged from top to bottom to guide you through the acquisition procedure There are four basic steps to scanning an image using the GS 700 1 Select the application 2 Select the scan area 3 Select the resolution of the scan 4 Acquire the image D 2 Step Select Application To set the parameters for your particular scan you can 1 Select from a list of possible applications or 2 Choose your own filter and light source settings Selecting from the List of Applications When you select from the list of applications the software automatically sets the appropriate filter s and other parameters for that particular application To select from the list of applications click on the Select button under Select Application ray film Coomassie blue Silver stain Copper stain Zinc stain Blot Stain all HEP Subestrat Fig D 3 Example of the application tree in the GS 700 dialog box Appendix D GS 700 The applications are listed in a tree that expands from left to right First you select the category of your application then you select your particular application To exit the tree without selecting press the ESC key
465. the name of the application You can also use this feature to create a new custom application from an existing one You can delete a custom application by selecting Custom Delete and the name of the application K 3 Step Il Position Focus Before positioning and focusing the image select the application that you will be using under Step I Note that the optimal position and focus will be different for a filtered image versus an unfiltered one All standard applications except Chemiluminescence use a filter Position After you have selected your application you are ready to center your gel or other object within the camera frame To do so click on the Position button The VersaDoc will begin capturing a live image and updating it every second With the Position button selected study the image in the acquisition window while you position your object in the center of the sample stage If you have a zoom lens on the camera you can adjust the magnification while you position See the VersaDoc User Manual for details on positioning While you are positioning you can select the Show Alignment Grid checkbox to display a target grid overlay on the image When you click on Position the light inside the camera box automatically switches on To turn the light off while positioning deselect the Light On checkbox When you are finished positioning click on the Stop button Focus Note Before focusing you should adjust th
466. then selecting a gel from the drop down list e You can print spot annotations by clicking the Annotation button and selecting from the list of annotation categories The report will include annotation information for the spots that have annotation data in the selected category Other Options Data Choose whether to round values off the the closest integer or include noralized values Missing Spots You can choose to estimate missing spots exclude them from the report or enter a minimum value to include in the report Saturated spots You can choose to estimate saturated spots exclude them from the report or enter a minimum value to include in the report 11 10 11 Graphs and Reports Once you have selected your options click Done to open the report viewer Ll Quantity Table Report HalfStock En E Ioj xj s8_ stockt stock2 stocks ate naire hat mz ser sol sro sa tal 13 Reformat report ao sax Page E of 62 gt e Fig 11 10 Viewing a Quantity Table Report The report viewer allows you the option of printing or exporting the report for use in a spreadsheet application Click reformat report button to return to the Quantity Table Report dialog You can also click on a row to highlight the spot in the MatchSet 4a Quantity Graph Report If you select Quantity Graph Report from the Report menu a dialog box will open in which you can specify the format of your quantitation histograms 11 1
467. this feature deselect the Auto All Same checkbox under Display Preferences Edit gt Preferences Note Shift F4 Hide All Overlays will conceal overlays in all subwindows regardless of whether Auto All Same is selected Viewing and Editing Images 3 8 Density Tools The tools under the View gt View Density submenu and on the Density Tools toolbar are designed for advanced users when analyzing gels They provide a quick measure of the signal intensity of the data in Raw 2 D scans or Filtered images Note that these commands will not work on Gaussian images MEE te E Plot Density Density at Cursor O Densitvin Box Show L d Bard Ne A eee Plot Cross section Cl T Ee OES 2 Plot Density Distribution Plot Vertical Trace Fig 3 6 Density submenu and toolbar Density at Cursor To display the signal intensity at a point on the image select Density at Cursor from the submenu or toolbar and click the image The pop up box will list the intensity in image units ODs or counts and will also show the average intensity for a 3 x 3 image pixel box centered on that point F Density in Box To display the total signal intensity inside a specified region on the image da select Density in Box from the submenu or toolbar and drag a box around E the area you want to measure The area of the box in square millimeters the total image intensity volume inside the box in image units ODs or counts and the av
468. this procedure can resolve complex biological samples with a high degree of specificity and accuracy The resolved proteins and polypeptide fractions can be then identified by their molecular weights and charges as indicated by their locations in the Raw 2 D gel as well as by their differential expression in different samples proximity to other spots intensity etc Raw 2 D electrophoresis involves two sequential separations of a sample in perpendicular directions The IEF dimension 1s run first in tube gels or on immobilized pH gradient IPG strips After focusing the tube gel or strip is placed on top of an SDS PAGE slab gel and electrophoresed This technique can resolve thousands of protein spots in a single sample these proteins can then be visualized by metabolic radiolabeling or a variety of staining methods Proteomics Applications Proteomics is the study of protein expression and regulation in cells tissues and entire organisms Several thousand proteins are expressed at any given moment in an organism at the cellular level dozens of proteins may be expressed and regulated in fractions of a second Two dimensional gel electrophoresis is a a cornerstone in the study of how proteins are expressed regulated and modified throughout living systems Developed almost a quarter of a century ago Raw 2 D gel technology remains one of the most powerful techniques for resolving complex mixtures of proteins The technology has improved
469. tic spots in the Gaussian image Note Any spots not detected in the Filtered image will not be included in the Gaussian image You can add spots to the Gaussian image during spot editing as described in following sections A Raw 2 D Scan Filtered image and Gaussian image of the same gel are part of the same scanset they all share the same root file name with different extensions Therefore performing spot detection on an original Raw 2 D scan named Proteins gsc would result in a Filtered image named Proteins gim and a Gaussian image named Proteins gsp After spot detection the Raw 2 D scan Filtered image and Gaussian image will be displayed in subwindows of the same window The file name and type of each image are displayed at the bottom of each subwindow 4 12 Detecting and Editing Spots samplel vl x Raw 2 D Image a sample vl Filtered Fig 4 7 Example of a Scanset Note Use the Assign tool and Configure Subwindows command on the main toolbar to arrange images in subwindows See section 3 1 Windows and Subwindows for more information about viewing commands What Are Gaussian Spots Fuzzy streaked or overlapping spots in a Raw 2 D gel can be difficult to accurately quantify Because the image profile of an ideal spot conforms to a Gaussian curve PDQuest uses Gaussian modeling to create ideal spots that can be easily identified and quantitated A Gaussian spot is a precise three dimensional representat
470. timated or specified as the way in which saturated spots and unmatched spots are reported The quantitation of saturated spots can be estimated reported as 3 0 or reported in some other way that you specify If you opt for the latter enter the value that should be reported in the Specify field If a spot is unmatched in one or more members of a MatchSet it can be reported as the minimum spot quantitation value the minimum quantity needed for a spot to be 2 32 General Operation detected for the gel Alternatively it can be reported as 1 0 or as a value that you enter in the text box next to the Specify button Next select the field separators you want to use to separate columns of data in your spreadsheets commas or tabs Your data can be exported as a file or to the clipboard If you choose the File option button clicking OK opens the Basic Export MatchSet dialog box in which you can enter a name and export location for the file If you choose the Clipboard option button clicking OK saves your data to the clipboard 2 6 Exporting Annotations You can export MatchSet annotations by category to a text file for inclusion in a document or spreadsheet With the MatchSet open select Export Annotations from the Report gt Export submenu The box prompts you to select the annotation category or categories to export 2 33 PDQuest User Guide LL Export Annotations ms A a xx Select Categories To Expo
471. ting Images Note If you are pasting into an image with a different pixel size 1 e resolution you will receive a message that the placement of the copy may not be exact Click OK to complete the paste then position the pasted objects manually Viewing Previously Created Text Overlays Lines To display previously created text overlays and or lines after opening an image click the Text Overlay Tools button on the main toolbar If you have concealed all your overlays using Hide Overlays clicking on any of the buttons on the Text Overlay Tools toolbar will display the hidden text 3 41 PDQuest User Guide 3 42 4 Detecting and Editing Spots This chapter describes how to detect and edit spots using PDQuest 4 1 Selecting Spot Detection Parameters The Spot Detection Wizard is used to select the parameters for detecting spots in gel scans Because gels vary in stain type spot intensity clarity streaking etc different parameters are needed to detect spots on different gels In general you can use the same parameters to detect spots on all gels that use the same stain and have been run and scanned in the same manner To select the detection parameters for a particular type of gel make sure that a representative gel scan is open and active in PDQuest then select Spot Detection Wizard from the Spots menu or quick guide PDQuest User Guide i Spot Detection Parameter Wizard Basic Advanced Step 1 Give Man
472. tions Display and uncheck the Create image analysis step option If the Create Image Analysis Step checkbox is unchecked the WorksBase explorer dialog will include the Experiment Step level just below the Experiment level so that you can select or create the desired experiment step This is most useful when you have created the experiment using a protocol in WorksBase for Proteomics and the step has been created for you N 13 PDQuest User Guide N 5 Saving Existing Files in WorksBase Open the image using the regular Open File dialog Invoke the Save in WorksBase command from the File menu The same dialog will appear that was described under Image Acquisition except that the name is not editable The new WorksBase file must have the same name as the original file on disk Select or create the Program Project Experiment Sample and Gel records to be associated with this image N 5 a Replacing existing items in WorksBase Note that you may not save a given image more than once in WorksBase The image was assigned an internal identifier when it was created and this is used to identify it PDQuest can recognize it even if you rename it If you try to save an image that is already in the database a warning message will appear and ask you to confirm the operation Appendix N WorksBase ll Confirm Overwrite The following tles are already in WorksB ase A If vou proceed these tiles will be replaced Do pou wish to
473. tipl Cuts OF he Sane Spol iaa its 8 14 CDP TAIN orcos ds 8 9 PAUSA AU ds 8 17 REUS DOS a Ea 8 19 SPEC Uh y mo CU rd pida eds 8 12 SCOP PHL A A O ede naa das 8 17 Basic Export Matchs8t crm ias 2 31 Bio Rad Technical Service A IAS 1 17 Boolean Analysis SUS nda tal 7 17 BOWS cAn S ia 7 33 Index 2 C Index Calibris ael SCANS eieaa N pida L 1 Calstrips Boxin ses menis mata aaae e aE E R L 7 CAUDA ON CURVE salio L 8 CoOntistrins MS WINDOWS sisas id L 2 CFOPPIN 2 OULOL NATES A A A L 1 Deactivatino a comentes ol als L 9 Encanta O o asas L 2 Geli OMIA ONE A ii L 4 Range Gl Vales aid il L 3 RECOLGS cnt L 2 REMOVE SEM o iio L 9 Sampline ses menis na ais L 6 Cancino DUES A ias 4 22 Catesones ot annotacions amaia alias 7 24 Centenne ima ES obte 3 9 Chemi Doc ACau ne AS IMA A cacas B 5 C 6 Adjustios ANG display salada oia ansia B 11 ATIMOLALIN imac ia iaci A 10 B 12 AUTO EXPOSE a casts actual a oad rcatiedentaniadnlcees B 6 C 6 AUO ePOS SCLIN 8 dd B 15 C 14 Backup Ia OCS toa Seeduinadseecansttedencuss B 16 C 14 A A A On B 5 BDAC Sena Sein a AE oeusdaucnalsia RATAA B 14 Exposure aS DAE Sera a E A T B 13 C 9 PE XPOSUPS Messiaen A 7 B 7 C 7 A eee at B 8 mane TMOG Ss A AN B 5 MAMA A a ES ica B 15 C 12 ver displasia B 11 LIVE AC UES a a ae E B 8 C 8 Eivelimasedisplay naa a os B 4 Manta EXPO aiii B 7 C 7 OPOS Amada dada ads B 13 C 11 Index 3 PDQuest User Guide Postionms Ee sample ii Ada B 4 C 5 Saturated
474. tivity see below When you select an application the software automatically sets the appropriate standard filter S20LP 530DF60 610LP clear or none light type UV white or none and light source trans epi or neither in the Fluor S for that particular application For applications involving trans illumination you must also specify a scan dimension see below Your selection will be displayed below the Select button To exit the tree without selecting press the ESC key G 4 Appendix G Fluor S Custom Applications If your application is not listed if you want to use a user installed filter or if you want to access High Sensitivity mode see below you can create and save your own custom application From the application tree select Custom then Create This will open a dialog box in which you can name your application and select your settings Mucleic Acid Gels Protein Gels Quantity One Blotting Create anew Fluors Application Name cel Type 44xc Densitometry Custom Filter User Falter 2 llumination White Epi Mode High Sensitivity Cancel Or Fig G 4 Creating a new custom application To select the filter including user defined type of illumination and camera mode click on the appropriate buttons and select from the pop up lists Note Under Illumination there is a listing for a spare UV light source This selects the spare UV bulb in the Fluor S Select th
475. to compare replicate gels or classes then choose which members to compare by clicking on the drop down buttons for A and B Indicate the statistical test you want to perform by clicking on the button next to the name of the test Use the radio buttons to refine the confidence level 7 13 PDQuest User Guide Refer to the following table to determine the number of gels per group for you to run a specific test Table 1 Statistic Analysis Types ea Maximum gt Minimum Minimum of Replicate of Classes of gels samples Groups sample per group per group or class N Mann Yes Yes 4 NA Whitney hosel i AA The Wilcoxon test is only available if you are comparing classes You must have at least six samples per class Follow these steps to create sample pairs in order to use Wilcoxon test A 20 25 class only Step 1 Create samples in sample database See Section 6 6 Sample Database Step 2 Create an attribute for these samples Enter values for this attribute Each attribute value pair defines a class For example an attribute could be treatment with values of treated or untreated This defines two classes treatment treated and treatment untreated See Section 6 6 Sample Database for further information on attributes Step 3 For the Wilcoxon test the two classes should have same number of samples and there should be at least 6 samples in each class Step 4 Pair the samples in the two classes See
476. to the original view Note Reset view does not change the scale factor To reset the scale factor close the 3D Viewer and click the box again to re open the 3D Viewer with the original scale factor 3 12 Transform The Transform controls are used to adjust image brightness and contrast and optimize the image display These controls affect the display only not the underlying data 3 20 Viewing and Editing Images Select Transform from the Image menu or main toolbar and click in the subwindow or use the Ctrl B key command in the subwindow Image Spots Match Analysis al gt L a gh ag Pi E in Transform Ctri B Crop Advanced Crap Li Wertical Flip Horizontal Flip Rotate Filter Wizard Filter List Invert Data Reduce File Size Fig 3 14 Transform command The preview window in the Transform dialog shows a smaller view of the selected image Changes are automatically reflected in the preview window and are only applied to the main image s when you click OK or PDQuest only Apply All 3 21 PDQuest User Guide Transform Plot Preview Window E Transform ahmed Master Modified the view of the da a nos the data diss Tranadorm Plot Frequency Distabubor f Fulscale Low High x Log High y 2498 image N y Display Lw m fira Max 35060 Invert display Log High Low Slides C ateated Quariiy raw Min fo Highlight sahsated pets Ahua subo scale
477. tog Browse X Defaut al MaseLynx Projects C Program Files ProteinLynx Global Serveridemodbs sprotlas_gs Browse Ba MaseLyns Install C Program Files ProteinLynx Global Servercornfa Browse C Mascot Install C Mascot Install Browse Mascot server UAL http fktroll mascot coi C Temporary CA Program Files Bio Rad The Discover Seres tmp WA DK X Cancel 1 Help Browse For Folder Browse x Default Select Directory Ey ProteinLynx Global perwer om Ess a config See demodbs ve sprot fas_gs eal jars Lime lane xl Folder sprot Fas_gs Cancel New Folder Pa Fig 2 24 Path preferences Browse button To set a particular file path click the Browse button next to the path field and navigate to the appropriate directory then highlight the folder where you want to set the path Click OK Check Default to return the path to the default setting The Images path Browse button connects to the main directory where you keep your image files 2 37 PDQuest User Guide The MatchSets path Browse button connects to the main directory where you keep your MatchSet files The Log path Browse button sets the directory where spot detection log files are saved Select Spot Detection Report from the Spots menu to access the log files in this directory 1 PDQUEST NE x il PDQUEST 9 Spot detection reports Auto Detect Status ETrof 20010906 1231 RTro11_20010906_1231 September 6 200
478. tool closes and the new configuration takes effect 11 1 a Adding and Editing Configurations To add a new configuration click Add or to edit an existing configuration highlight the desired configuration and click Edit This opens the Configure Graph dialog Graphs and Reports Ll Configure Graph 4 a x Name all Replicate Groups Description Quantification Mode Color Replicate group name f All Gels All Replicate Groups f All Classes ES Stock Ee Halt Display Mode Individual Group x Colorize yf Done X Cancel 7 Help Fig 11 3 Editing a Configuration Step 1 Enter a name and description in the requisit fields Step 2 Next select whether you want the graphs to show quantifications for gels groups or classes Note If you have not created any replicate groups or classes these choices will be Inactive Step 3 Select the display mode This allows you to either view quantifications for replicate groups and classes as an average of the spots in the group or class or as individual graphs for each spot Selecting colorize colorizes the graph based on the groups or classes This 1s very useful when viewing individual spots for replicate groups 11 3 PDQuest User Guide SSP 4203 SSP 4203 Individual Group Fig 11 4 Colorized graphs showing replicate group by individual and group To delete a configuration highlight the item you wish to delete and cl
479. tour tool ls E of iS y Scanset sample2 Y 2 EE Ez sample2 v2 Filtered Modified sample2 v2 xl Raw 2 D Image Modifi sample2 v2 Gaussian Modified hi Scanset sample2 10 x sample2 v2 Filtered Modified sample2 v2 x1 Raw 2 D Image Modif sample2 v2 Gaussian Modified 2 Drag a contour around the spot in the Filtered or Raw 2 D image 3 The contour is created and the spot is added to the Gaussian image Fig 4 13 Creating a spot contour To edit the boundary position your cursor on the border with the contour tool Your cursor will change to a pencil Drag across the line a new yellow line will appear The boundary will look blue when not selected yellow when it its being edited and green when it is selected Spot Freehand Tool To use this tool first magnify the spot you want to quantify you must be able to see the individual pixels in the image Then select the Spot Freehand Tool from the toolbar or submenu and click and drag around the edge of the spot to enclose it in a boundary When your line crosses itself a freehand boundary is created If you make a mistake while drawing simply backtrack with the mouse The line you draw should completely surround the spot you want to quantify 4 20 Detecting and Editing Spots Spot Boundary Tools Ea a 1 Select the freehand tool ale hi Scanset demol 2 Draw the freehand line around the spot in the Filtered
480. ts Raw 2 0 Images Zoom 10 0 f As single spot Pan 90 0 nee Gel Spot Bkgd lo 0l As multiple spots Filtered Images 7 Gaussian Images 2 Jump Cursor On Alert x Bold match symbols Auto Imitate Zocor _ Filled spot ellipses O Click to focus subwindow x Solid label background 4 Auto All Sanne x Small window labels x Save subwindow configuration wf DK X Cancel EJ Help Fig 2 26 Display Preferences Click the Display tab to access the following preferences Zoom allows you to specify the percentage by which an image moves closer or farther away when you use the Zoom functions This percentage is based on the size of the image Pan determines the percentage by which the image moves side to side or up and down when you use the arrow keys This percentage is based on the size of the image 2 39 PDQuest User Guide If you want to apply a uniform background to Gaussian images so they more closely resemble the original gels enter a background optical density O D value in the text box next to the Gel Spot Bkgd prompt The Default type option lists the three images associated with a MatchSet Select the image you wish to have displayed when you create or open a MatchSet Note If you have Save subwindow configuration selected this overrides your Default type selection when you open a MatchSet In Gaussian images specify whether you want to see only single spots
481. ts in the Analysis set Template check the box labeled Display Selected sets 7 1 d Quantitative Analysis Sets Quantitative analysis sets allow you to perform fold change analyses To create a Quantitative Analysis Set select Quantitative from the Select Set Type dialog 7 8 Analysis Sets and Annotations i Analysis Set Template Matchset HalfStock e i Ee E a Hame Quant Description Options Manan 2 Include qualitative changes Min told over background E E Compare e Gels 5 stock2 Repl Gels B harra _ Display selected sets f Classes 5 Mark spots xe Mark spots on Method f Above upper limit 7 Below lower limit Between limits Dutside limits 2 00 2 0 fold e Master gel Operand gels All gels Show a Window labels J55P number Format 1 0 Spot count s6s e Histogram 0 50 2 0 fold f Scatterplot x Log scale A E Upper lirnit e 2 00 Lower lirnit os o 50 1 upper yf Save X Cancel 2 Help Fig 7 4 Quantitation Analysis Set dialog Enter a name and description of the analysis set 7 9 PDQuest User Guide Note If you have Normalization enabled normalization parameters will be used for spot quantities See Section 6 7 Normalization for further information Quantitative Analysis Set Options The following options help refine the analysis set Estimate missing spots Otherwise the spot wi
482. tting if you are using gels that have a backing on them The Tip motion range determines the side to side motion in millimeters of the tip as it is cutting backed gels The default range is 2 0 while the maximum range is 5 0 mm The user should determine the optimum setting for their gel Note The Backed gel option is only available if you are using the ProteomeWorks Plus spot cutter Note The backed gel option is recommended for the 1 0 mm cutting tip only Use of the 1 5 mm cutting tip may result in decreased gel pickup efficiency The Gel Defaults button automatically selects the most useful options when cutting gels The PVDF Defaults button automatically selects the most useful options when cutting PVDF membranes Select the wash stations to use after each cut by clicking on one or more checkboxes Station 1 Station 2 etc The wash solution is set by the user Typically water is enough for washing Under Misc Options select Multiple Cuts Spot to specify multiple cuts of larger spots The number of cuts spot is selected in the Excision Gel Selection window Note that the software will automatically calculate the size of each spot based on its Gaussian profile in the MatchSet gel when determining whether to make multiple cuts Save Validation Image will create and save an image of the gel on the spot cutter platform when the cut run is complete You will be prompted to name the validation image Step 3 Select the Microtiter P
483. turned on the average quantity of that spot in each replicate group will be displayed as will the coefficient of variance CV and the number of gels in the replicate group Count 4 11 Spot Quality Spot quality is a number ranging from 0 to 100 that is calculated based on five attributes of each spot 4 26 Detecting and Editing Spots e Gaussian fit PDQuest determines how well the spot fits the Gaussian model and assigns a value based on this fit e X streaking The software determines how much of the spot is affected by gel streaking in the x direction e Y streaking The software determines how much of the spot is affected by gel streaking in the y direction e Overlap The software determines the percentage of the spot that overlaps with another spot or spots e Linear range of scanner PDQuest determines whether the peak intensity value of the spot is within the linear range of the scanner Each spot attribute is evaluated and weighted to produce a single numerical value for Spot Quality If a spot fits the Gaussian model perfectly has no streaking in the x or y direction does not overlap with any other spot and has a peak intensity within the linear range of the scanner PDQuest will assign a value of 100 to the spot To display the quality of all the spots in a single image select Show Quality from the Spots menu and click the image To display the quality of all the spots in all the subwindows of a selected window s
484. ual Guidance Ql Click on a faint spot Ce Click on a small spot if different from the faint spot LA Box the largest spot cluster Step 2 Test Settings Sensitivity o 67 el Size scale 3 tl SP Find Spot Centers Min pek s1s0 e Spot Count o _ Gaussian model during test takes longer Help Optional Controls Streaks Background Smoothing Speckles Vertical Radus ss Horizontal Radius las el Large spot size Help ty Be ee Slee Image samplet v1 x1 Raw 2 D Image Process All Gels X Quit E Help Parameter Set Matts par Load Save Fig 4 1 Spot Detection Parameter Wizard The Wizard will open and the name of the scan will be displayed below the preview window The parameter set field indicates the name of the currently loaded parameter set If no set is loaded this field is blank Click on the Load button next to the parameter set name to load a different set of parameters into the Wizard You can choose between the Basic and the Advanced tabs With the Basic tab you can perform Steps 1 and 2 of the parameter selection process The Advanced tab offers the addition of Optional Controls to manipulate streaks backgrounds and speckles 4 2 Detecting and Editing Spots Image 18237 vl x3 2D Scan y i PDQUEST ES A Select scan to analyze Bean 10 54 days Scan Scan Scan Scan Scan Scan Scan Scan Fig 4 2 Selecting
485. ulated from the spot intensity in the image 7 fla To make a single cut in a spot make sure the Multiple Cuts Spot checkbox a is unchecked then click the Add Cut button and click on the spot in the image that you want to cut hi Basic Excision Tool Step 1 Acquire Image Light on Gel Light off FDF Acquire Image Uy on Fluor Time secs 3 000 Step 2 Specify O iii Fe Add cut T Remove cut X Multiple cuts sp Cuts 2 E Max cuts spot E Excisions 2 x Snap to peak Step 3 Set Cutter Options X Spin when cutting _ Cut longer X Spin when ejecting X Double eject No spin when lifting JBacked gel Tip motion range 2 0 Gel defaults PYDF defaults Step 4 Set Wash Options O Station 1 x Station 2 X Station 3 X Station 4 isol bosib Q Begin Resume r3 aana Display X Well number _ Cut position Setup Save Confirm cuts E Re cuts EM Close 4 Help Hua 00 Simulate X Cutter xX Camera Fig 8 7 Adding cuts 8 12 Basic Excision Tool If this is the first cut added the Enter Plate Information dialog box will open first when all the pertinent information is added the cut will be added to the image Gi Enter Plate Information Hame Plateool Loading order l AZ AS Starting well A01 Wells skipped to yf Done Cancel Fig 8 8 Enter Plate Information dialog Enter
486. um slider defines the pixel value that will appear as white in the image while the maximum slider defines the pixel value that will appear as black The slider scale is 0 255 with the defaults set to 60 minimum and 130 maximum Imaging Area These fields are used to specify the size of your imaging area in centimeters which in turns determines the size of the pixels in your image 1 e resolution When you adjust one imaging area dimension the other dimension will change to maintain the aspect ratio of the camera lens Note Your imaging area settings must be correct if you want to do 1 1 printing These are also important if you are comparing the size of objects e g using the Volume Tools between images Auto Exposure Threshold When you click on Auto Expose the exposure time is determined by the percentage of saturated pixels you want in your image This field allows you to specify that percentage Typically you will want less than percent of the pixels in your image saturated Consequently the default value for this field is 0 15 percent Reminder When this checkbox is selected the software will warn you to turn off your transilluminator light when you exit the ChemiDoc acquisition window or when your system is idle for more than 5 minutes Note If you are performing experiments that are longer than 5 minutes e g chemiluminescence this should be deselected Video Printing Footer Information The checkbox
487. unt 5 O Drag items to reorder them Step 3 Create MatchSet Name Description Comments Directory C 1Program Files Bio Rad The Discovery Series Sampl Char A Member sharing Do not make copies refer to the originals Copy the originals into this matchset s data folder JDisable this step Step 4 Match Gels Step 2 Detect Spots on Gels Parameters samples Edit Parameters De fault Match Settings gt Edit C Disable this step Log File Name KTro11_20011010_1122 3 Go X Cancel u PDQUEST Auto Detect Parameters samplel xl sample3 samples samp leahm scansetZ scansetZ2l Cancel Fig 5 6 Automated Detection and Matching dialog When you have completed Step 1 Select Gels to Analyze and Step 2 Detect spots you are ready to proceed to Step 3 Create MatchSet Follow the same procedure as you did when you Created a New MatchSet as far as Name Description Comments and Member Sharing If you want to change the default directory in which you store your MatchSets click Change To select the directory in which the MatchSet will be saved click Edit gt Preferences gt Paths tab browse to select the appropriate directory folder and click OK Otherwise if the default button in the Paths tab is checked the directory defaults to the last directory you used 5 8 Matchsets If you only want
488. up your four most common applications using the different channels but only enable Channel 4 for a particular gel Channel 1 would be scanned first Channel 4 second PDQuest User Guide Enabled channels are imaged sequentially The separate scans are displayed and saved as separate images The total imaging time depends on the number and type of enabled applications If you have selected Auto Save After Scan the image created using Channel 1 will have the base file name and images created using subsequent channels will have the base file name plus a version number v 2 v 3 v 4 Note that the image version number does not necessarily correspond to the channel number For example if you captured an image using only Channels 1 and 4 the image created using Channel 4 will be saved as version 2 v 2 K 2 b Selecting an Application To select an application i e the appropriate filter and illumination source for the type of object you are imaging click on the Select button under a channel tab Standard Applications The standard applications and associated settings are listed in a tree that expands from left to right When you select an application the software automatically sets the appropriate filter in the VersaDoc for that particular application First select your general application then select the particular stain or medium you are using When you select the stain or medium the software automatically sets the appropria
489. ure nimes TeCOMMEndSd ia ab G 9 ASA AA A a AR C 15 G 17 PP santos deen aoaaaceneouetcod sat oaaeinds uiagasadaniaashinaimiaessdniunesenspeimonisaneinces G 7 High resolution high sensitivity tdi G 6 DU OUS aa ooo riloho pio il Eds G 11 POSI ONIS a ito to iaa cos aba G 6 A SCA aea e a aaa EEEE EN E SE D G 9 Saturated pixels highlighting ooooooonnonnnnonoonnnnnnnnnnnnononnonononnnonnos C 15 G 17 Cal GME NSI ON iia illinois G 6 DElSCUNS AN AP PlICAUON seprana na a AET G 4 STU PAM ONTO ul aero E N Eas G 2 Index 6 Index FX see Molecular Imager FX G CAUSA A a 4 12 GAUSSIAN POS aia 4 13 Gel Doc POC QUIT SONS TIN ACO os A 5 O A 5 AUO EXPOSUTE SEUUINOS naaa di nays A 13 Backup mares ita A 13 DAC SONOS aa a O A 12 Display sena S a E ia A 8 Exposmie Solais Dar ani A 11 POD E EAE toad P E T E E N I E A A T TA EAA A 7 TASS mode aa a S A 5 A A 13 TY CLS DIAY aaron cade A 9 Dive imate display a e A 4 Manuale pose arena a Seth Miandad aula enatiedeabeasiacnlcaacsatedens A 6 OPCON sea E E A 11 Posti nme he sample sacri a A 4 Saturated pixels melo A 9 SAVNO Ima ad a a A 11 Simulation Md io A 2 UY Mod siste taetoes A 5 ACC OSC A A 2 Video display wimdOw aa fast arnt T A 3 Video Prntins Tooter MO as A 13 Wihite e htmoders insano decos A 5 CHEW IREC ONG a a a aa L 4 CTA Sahih E A Dscamnndty aicacebaadecanect E eamscaeaaeate 3 8 Graph ANAIS dra 11 7 Conmara e ARRANCA S A N 11 7 Marsin rapi uns A 11 6 Index 7 PDQues
490. ure time in seconds directly in the field or use the Arrow buttons to adjust the exposure time in 10 percent increments Step Ill Set Exposure Time sec PRE e gt Preview Fig K 5 Selecting an exposure time Appendix K VersaDoc The following table provides recommended exposure times for various applications Table 1 Recommended Exposure Times and Lenses Recommended Sample Exposure Lens amp Filter Accessories Used Fluorescent Stain Gel Zoom R Fluorescence End 30 sec 5 min Zoom R None Label Gel Fluorescent Blot 0 5 5 sec Zoom IR Sample Chemi Tray if sample is small Chemifluorescent 0 5 5 sec Zoom IR None Blot Colorimetric Gel 0 1 1 sec Zoom IR White light con version screen Colorimetric Blot 0 1 1 sec Zoom IR Sample Chemi Tray if sample is small X ray film 0 1 1 sec Zoom IR White light con version screen Weak 5 10 min 50 mm Sample Chemi Chemiluminescence Tray if sample is small strong 10 sec 2 min Sample Chemi Chemiluminescence Tray if sample is small lFor chemi applications the 50mm lens is recommended Always remove the 660 fil ter K 9 PDQuest User Guide Note For most applications you can select an exposure time capture an image study it and then adjust the exposure time accordingly Repeat this procedure as many times as necessary to obtain a good image For chemiluminescent samples which degrade over time and emit low levels of light
491. ure time you selected is divided by the number of exposure counts set in the Fluor S Options dialog box see Options below For example if you enter an exposure time of 10 minutes and an exposure count of 20 then 20 intermediate exposures will be produced at 30 second intervals Each intermediate exposure will be displayed in the scan window The final full exposure will be displayed in a separate image window Note The first intermediate exposure will take longer than 30 seconds to display if dark subtraction is performed You can automatically save your intermediate exposures as separate files for later review using the Auto Save After Scan option See Options below If you see an intermediate exposure that you like in the scan window click on the Stop button Live Acquire mode will end and the last intermediate exposure to be completed will open in a separate image window You can then save it for analysis G 6 Options Click on the Options button to open the Fluor S Options dialog box G 11 PDQuest User Guide Fluor 5 Options Fluor S Information Dark Subtraction Type f Normal f Referenced None Reset Reference Live Exposure Mode Exposure Court s mnsan Fluor 5 Multilmager simulated Imager Firmware version 1 01 Imager Hardware Version 1 01 Imaging Area cm Width 13 17 Ch Height 10 35 Chi Save Hel _JAuto Save After Scan x Auto scale Transtorm After Acquistion
492. ures Highlight Saturated Pixels When this box is checked any saturated pixels in the image will appear highlighted in red in the scan window and in the pop up image window To view hide saturated pixels in the pop up image window use the Image gt Transform command H 16 Appendix H Fluor S MAX File Size of Images Image File Size below Options shows the size of the image file you are about to create This size is determined by whether the image was created in High Sensitivity or Ultra Sensitivity mode If you do not have enough computer memory for the specified file size an error message will appear when you attempt to acquire an image Macintosh users can increase the application memory partition See your Macintosh computer documentation for guidance PDQuest User Guide H 18 Appendix Personal Molecular Imager FX Fig I 1 Personal Molecular Imager FX Before you can begin acquiring images using the Personal Molecular Imager FX the instrument must be properly installed and connected with the host computer See the Personal FX hardware manual for installation startup and operating instructions Note The Personal FX should be turned on and the initialization sequence completed before the host computer is turned on except in the case of certain Power Macintosh configurations See the hardware manual for more details PDQuest User Guide PC Only A Note About SCSI Cards The Personal FX is connected to
493. ussian mpdil STD mp di00318 std Q 3894 vO x0 Ganssian mpdi00314 3679 v0 x0 Ganssian mpdi0031 3553 v0 x0 Gaussian mpdi 3690 v0 x0 Gaussian mmpdil 4411 vO x0 Gaussian mpdi003199 3897 vO x0 Gaussian mpdi00314 ii HL Matchset MPDI com arison The masters of the two level 1 MatchSets be come the members of the higher level match sets STD MPDI comparison Jstd mpd MPDI comparison mpdi00137 5td H MPDI comparison mpdi00313 5td Fig 5 20 Example of a higher level MatchSet Creating a Higher level MatchSet Use the Create Matchset dialog Match gt New MatchSet to create Higher level MatchSets the same way you created Level 1 MatchSets Under MatchSet level select the radio button for Higher level MatchSets Add entire MatchSets as members instead of individual gels Higher level sets are similar to Level 1 MatchSets see section 5 1 Creating a MatchSet except 1 You select the Higher Level option button instead of Level 1 2 You select MatchSets as members instead of images Note that you cannot include Filtered images in a higher level MatchSet because higher level MatchSets are made up of Masters which do not have Filtered images Because higher level MatchSets are linked to the lower Level MatchSets they are created from they should not be moved from the file directory in which they are created If you want to create a higher level MatchSet to be shared among a group of 9 35 PDQuest Use
494. variations in the size and or intensity of a particular spot will be averaged out when the group of spots is considered is a whole Total Quantity in Valid Spots In this normalization method the raw quantity of each spot in a member gel is divided by the total quantity of all the spots in that gel that have been included in the Master see the normalization formula Select the Total Quantity in Valid Spots option button in the Normalization dialog to activate this method This normalization method assumes that few protein spots change within the experiment and that the changes average out across the whole gel It can be useful if very little information is known about the possible source of sample variation Total Density in Gel Image In this method the raw quantity of each spot in a member gel is divided by the total intensity value of all the pixels in the image see the normalization formula Select the Total Density in Gel Image option button to activate this normalization method This model assumes that the total density of an image 1 e background density plus spot density will be relatively consistent from gel to gel It can be useful if very little information is known about the possible source of sample variation Specified Value In this method the raw quantity of each spot in a member gel is divided by a user defined normalization factor for that gel Select the Specified Value option and enter a normalization value f
495. ve As from the File menu Analyze Clicking on Analyze will open a separate image window displaying the captured image The default name for the image will include the date time and user if known You can then analyze the image using the other features in the main application The image will not be saved until you select Save or Save As from the File menu A 7 Step VI Select Output In Select Output you can select Video Print and Save as your output options Step Yl Select Output Video Print Save Fig A 8 Output options Video Print Clicking on Video Print will automatically send the currently displayed frame either live or integrated to a video printer You can add information about your image to the A 10 Appendix A Gel Doc bottom of the printout by selecting the appropriate checkboxes in the Options dialog box See section A 9 Options save Clicking on Save will open a separate image window displaying the captured image A Save As dialog box will automatically open displaying the default file name for the image which will include the date time and user if known You can then change the file name and storage directory You can also export your image as a TIFF image for viewing with other applications A 8 Exposure Status The Exposure Status bar shows the progress of your exposure If your exposure time is greater than 1 second the status bar display will give you a graphical represen
496. ve mode is for scanning transparent mediums such as films gels and slides The scanning dimensions in this mode are 20 cm X 25 cm uncalibrated D 3 Step Il Select Scan Area Preview Scan Before selecting the particular area to scan you can preview the entire scanning area to determine the exact position of your sample Click on Preview Scan A quick low resolution scan of the entire scanning area will appear in the scanning window Your sample should be visible within a portion of this scan D 6 Appendix D GS 700 Selecting an Area Using the preview scan as a guide select your scan area by dragging your mouse within the scanning window The border of the scan area you are selecting will be marked by a frame Note The scan area you select must be at least 1 cm wide When you release the mouse button the border changes to a dashed blue line indicating a selected area e To reposition the scanning area box you have created position your cursor inside the box and drag The entire box will move e To resize the box position your cursor on a box side and drag The side you have selected will move e To redo the box entirely position your cursor outside the box and drag The old box will disappear and a new box will be created You can also select the scanning area by entering coordinates in the appropriate fields Top Bottom Left Right As you enter a coordinate the position of the scanning area box will change
497. vel of the mass spec protein identification Select Mass Spec Score Overlay from the Identification menu 10 5 PDQuest User Guide Lh Mass Spec Score Overlay 3 Instrument f MALDI MS MS Bazsedon e Jont probability MOWSE Threshold o_o Above threshold Format Below threshold Format Mo hits Format yf Done H Help Fig 10 2 Mass Spec Score Overlay dialog In the dialog box indicate the instrument you want to report scores for by selecting MALDI or MS MS Note that if there is no data for an instrument no spot overlays will be shown Next select the type of score by selecting Joint Probability or MOWSE Enter the threshold value for the score in the Threshold field Spots above this threshold can be considered confident hits spots below this threshold are not Click the Format buttons next to Above Threshold Below Threshold and No Hits to select the appearance of the different overlays No Hits are spots with no hits or with an unknown protein identification This means that the mass spec tried and failed to recognize the sequence 10 4 Protein Probe If you have imported mass spec data for spots using Import MassLynx Results you can launch the Protein Probe browser from within PDQuest to review the protein information for those spots Note Protein Probe must be installed on the same computer as PDQuest to use this tool 10 6 Mass Spectrometry Analysis With you
498. w Program level dd mm ypuy _ Programm is required mari x Show Project level CO ppyymmdd e 01 Jan 2001 01 Jan 01 _ Project is required JS how Image level x Sutomatically generate Image Analysis Experiment Step Show Identifiers in lists yf Done X Cancel 2 Help Fig N 5 WorksBase Options Display tab Show Program level The use of Programs is optional If you do not want to use programs this provides a means to hide the Program level on the Explorer Program is required If you select Program is required then images may not be saved in WorksBase unless a Program is specified Show Project level The use of projects is optional If you do not want to use projects this provides a means to hide the Project level on the Explorer Project is required If you select Project is required then images may not be saved in WorksBase unless a Project is specified PDQuest User Guide Show Image level Most users will select images to load by choosing samples or gels Hiding the Image level reduces the complexity of the Open dialog Automatically generate Image Analysis Experiment Step Select this option to have PDQuest automatically create the Image Analysis step whenever you save images or MatchSets in WorksBase Show identifiers in lists This shows the internal identifiers in selection lists These are large and cryptic so are not normally wanted However this can be
499. will be the final exposure If you did not elect to auto save the exposures as they were created then each image will be unsaved To stop the Live Acquire click on the Freeze button Note Exposures captured before freezing will be displayed in image windows Study the different images and select the best exposure s to keep C 4 a Exposure Status The Exposure Status bar shows the progress of your exposure If your exposure time is greater than 1 second the status bar display will give you a graphical representation of the remaining time before exposure is complete Illumination Flat Fielding For UV or white light transilluminatoin applications you should use the appropriate reference plate to ensure a uniform intensity in the image This will compensate for normal variations in image pixel intensity that occur with a transilluminating light source C 9 PDQuest User Guide To enable this feature select the Flat Fielding checkbox UV Illumination Flat Fielding When you first select the Flat fielding checkbox and then acquire an image using the UV transilluminator you will be prompted to remove your sample and place the fluorescent reference plate on the ChemiDoc XRS sample stage and turn on the UV transilluminator see the ChemiDoc XRS User Manual Quantity One xx AN lurmination Flat Fielding Remove your sample fromthe ChemiDoc ARS Continue Cancel Fig C 9 Illumination flat fielding reminder
500. with the Edit button However unlike Spot Detection parameters the matching parameters work over a wide range of images and you will probably obtain satisfactory results with the default parameters Note Note If you want to detect spots without creating a MatchSet check the Disable this step checkbox at the bottom of Step 4 spot Detection Report To review the log file created during spot detection select Spot Detection Report from the Spots menu This report documents when spot detection began which gels were detected or failed to detect which steps were performed and when spot detection was completed PDQuest User Guide 4 3 Filtered and Gaussian Images When spots are detected in PDQuest the original gel scan is filtered and smoothed to clarify the spots then three dimensional Gaussian spots are created from the clarified spots The end result is three separate images the original Raw 2 D scan which remains unchanged the Filtered image which is a copy of the original scan that has been filtered and processed and the Gaussian image which is a synthetic image containing the Gaussian spots Gel image Gaussian Image Fig 4 6 Images created during spot detection All quantitation and other analysis are performed on the Gaussian image PDQuest preserves all three images so that at any point you can go back and reexamine the original scan and the Filtered image to compare the original spots with the synthe
501. without the motorized zoom lens Step Position Gel D Live Focus IRIS DOM FOCUS 4 4 aj a Show Alignment Grid Fig A 3 Newer Gel Docs feature camera control buttons in the acquisition window You can also select the Show Alignment Grid checkbox to facilitate positioning Note After positioning your sample you should check the Imaging Area dimensions under Options see section A 9 Options to make sure that they conform to the size of the area you are focusing on To determine the size of the area you are A 4 Appendix A Gel Doc focusing on you can place a ruler in the Gel Doc cabinet so that it is visible in the image A 3 Step Il Select Image Mode The Image Mode option buttons allow you to set the type and scale of your data UV Select this mode for fluorescent samples With this mode selected the data will be measured in linear intensity units White Light Select this mode for reflective and transmissive samples With this mode selected the data will be measured in uncalibrated optical density UOD units A 4 Step Ill Acquire Image For many white light applications you can skip this step and save and print images directly from Live Focus mode For UV light or faint samples you can take an automatic exposure based on the number of saturated pixels in the image or you can enter a specific exposure time Note Exposure refers to the integration of the image on the camera CCD
502. xes 8 2 a Step 1 Acquiring the Image Before acquiring an image select the light option appropriate for your gel membrane stain type e Light on gels e Light off PVDF membranes Ambient light will produce a better image for PVDF membranes e UV on fluorescent gels UV light is only available for the Fluorescent spot cutters PDQuest User Guide Step 1 Acquire Image Light on Gel Light off PDF Acquire Image UY on Fluor Time secs zs ood Fig 8 5 Light settings If UV light is selected specify an exposure time in seconds in the field Also make sure that the UV lamp switch on the back of the cutter box is on Note For Sypro Ruby stains an exposure time of 20 30 seconds is recommended When you have selected a light option click Acquire Image Note For UV exposures there will be a 15 second delay while the lamps heat up before the exposure begins The camera will take a single image of the gel or membrane on the platform then a background image to adjust for image background When the exposure is complete the image will appear in the window y To adjust the brightness and contrast of the image open the Transform window by clicking on the button to the right of the image window Y Tred Ske gee thes wet Bar 2 0 Image Basic Excision Tool Bota chit bruudsnn spandis change only he weee of the dibi cert he dila heel Transhom Plot Fieger Criintaaheon Fal sees
503. xposure time in seconds Reset Reference Dark Image Options Dark Exposure Time 30 oo Seconds OF Cancel Fig K 9 Reset Reference Dark pop up box Click on OK to implement your change The new reference dark will be created when you acquire your next image Because of the high sensitivity of the CCD fluctuations in background radiation and or temperature in the room can affect the level of dark count If you feel that radiation temperature conditions have changed in the room since your last reference dark was created use the Reset Reference button to delete your old reference and create a new one under current conditions None If you do not want to perform dark subtraction select None No dark exposure will be acquired or subtracted K 16 Appendix K VersaDoc K 6 b Save Auto Save After Scan To automatically save any image you acquire using the Acquire button click on the Auto Save After Scan checkbox in the Options dialog box With this checkbox selected when you click on Acquire a Save As dialog box will open asking you to specify a file name and location for the image you are about to create The scan will begin when you click on the Save button Make Backup Copy You can automatically create a backup copy of any scan you create To do so first select Auto Save After Scan then select the Make Backup Copy checkbox With this checkbox selected when you save a scan a backup copy will be placed in
504. y clicking on COM 1 or COM 2 9 2 6 Spot Cutter Calibration Before you can begin excising spots you must calibrate the spot cutter This procedure requires a coomassie blue stained PVDF membrane Note Before calibration you should focus the cutter camera and align the wash station position You should make sure the spot cutter camera image is properly set using the Crop Left Margin setting Note Ifyou are using the lens filter for UV light make sure that it is installed BEFORE calibration If you add or remove the lens filter after calibration you must recalibrate Click the Calibrate Cutter button A series of dialog boxes will guide you 7 through the calibration procedure The first dialog box will prompt you to specify whether you have installed the lens filter or not The lens filter must be INSTALLED if you are using the fluorescent spot cutter in fluorescent mode The lens filter is not required if you are using the spot cutter only in visible light mode 9 15 PDQuest User Guide Step 1 Step 2 Step 3 The second dialog box prompt for flat field calibration requires that you ensure the stage is completely empty and dry This includes the cutting mat which must be removed Press Ready to proceed If you have previously performed this step you can skip to Step 2 although this is not recommended i PDQUEST E xj Spot Cutter Calibration step 2 Image Field Calibration This step requires the calibration t
505. y problem e Grossly nonparallel lines in local regions e Lines of unequal length in local regions e Areas of the gel with few offsets this indicates unmatched regions 5 22 Matchsets hi Matchset rad matchset Modified Match symbols matched 13 unmatched Match offsets STD rad matchset std halfl Modified stocki v1 Filtered Fig 5 13 Vector offsets indicate that these two spots have been mismatched 5 6 Cybergels PDQuest needs to know the pH gradient of a gel in order to calculate isoelectric point values A new feature in this release allows you to combine overlapping areas of gels and create Cybergels Cybergels are a combination of gels with different pH gradients into a larger gel To create a cybergel select Combine overlapping gels from the Match menu Additionally you can select Match gt Automated Matching gt Primary Matching tab Then check the Enable overlapped pH range matching box to create a cybergel For example in the illustration below if the pH gradient set in the lab only goes to 4 you will only be able to detect proteins that separate at the pH level of 4 Due to the narrow set of pH ranges from 3 5 etc spots 1 2 and 3 are the same in gel 1 as in gel 2 And spots 4 5 and 6 overlap from gels 2 and 3 To create a cybergel eliminating overlapping spots or spots that occur in more than one gel you would add spots 1 2 and 3 from Gel 1 to the Cybergel and add spots 4 5 and 6
506. y selecting the 8 bit option Note Note TIFF images are exported from the Multi channel Viewer in 24 bit RGB mode to preserve the colors displayed in the viewer Finally if you are only displaying part of the image due to magnification or repositioning you can preserve the Current view or export the Entire image Exporting the Image The size of the pixels in the image and the file size of the image are listed at the bottom of the dialog When you are ready to export click the Export button A Save In dialog box will open The default file name will have a tif extension and the file type will indicate that this is a TIFF image You can change the file name or select a different directory to save in 2 3 d Closing files To unload a single image from a window or subwindow select the window or subwindow then select File gt Unload If the image is in a subwindow the image will close but the larger window will remain open if the image is in its own window the entire window will close If the file has changed since it was last saved you will be prompted to save the changes before closing The File gt Unload option is inactivated 2 15 PDQuest User Guide if this action is not executable If the action is executable you will be prompted to confirm File gt Close Alt F4 closes the entire active window including any subwindows If any of the images in the window has changed you will be prompted to save the changes before
507. yzing the spots in an experiment It contains copies of the Gaussian and Filtered images as well as the original 2 D scan of the gel s in an experiment Experiment MatchSet Master Note Gel 1 Gel 2 Gel 3 Image Image Image Master ES Fig 5 1 How experiments are organized into MatchSets If the Copy option is specified when creating the MatchSet see section 5 1 b Member Sharing 9 1 PDQuest User Guide A MatchSet can consist of one gel or many gels depending on the type and size of the experiment The MatchSet is displayed in a single window with subwindows for the images of the member gels and a subwindow for the MatchSet Master The Master is a synthetic image that contains the spot data from all the gels in the MatchSet All spot comparisons and analysis are coordinated through the Master Masters from different MatchSets can be grouped into higher level MatchSets for further comparison see section 5 8 b Higher Level MatchSets Using a MatchSet you can make quantitative and qualitative spot comparisons across gels calculate molecular weight isoelectric point values annotate spots group them into analysis sets combine the data from multiple gels into replicate groups and select spots for excision and mass spec analysis You can also use a MatchSet to combine gels with different pH gradients into a larger cybergel See 5 6 Cybergels hl Matchset MSO0004 7001 09 06 I sampled vl x1
508. ze After Height Y Pixel Size 300 00 by 300 00 microns Pixel Count 766 by 766 Memory Size 573 00 Eb OF Cancel Help Pixel Count and Memory Size reduced Fig 2 12 Reduce File Size dialog box before and after pixel size increase Note Asymmetric pixel reduction 1 e making pixels smaller in one dimension than the other is not recommended for 2 D gels since you want to resolve spots in both dimensions A pop up box prompts you to confirm the resolution reduction If you choose to reduce in place the descriptions of objects in the scan will be lost Only the scan image is retained Click Reduce and Copy to make a copy the original file and reduce the copy s size or click Reduce to reduce the file size of the displayed image Reducing the file size is an irreversible process For that reason we suggest you make a copy of the image and reduce its file size That way if you lose too much resolution you can simply delete the copy and try again Once you are happy with the reduced image don t forget to delete the original The goal is to save space 2 19 PDQuest User Guide If you choose to make a copy of the image you will be asked to enter a name for the new copy before the operation is performed 2 3 1 Exiting PDQuest File gt Exit quits the application You will be prompted to save any unsaved changes to images or MatchSets 2 4 Printing and Exporting The commands for printing images actu

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