Home

Overview of Melanie II 2-D PAGE - Bio-Rad

1. Hide Labels option If several features hold the same label AC accession number then by default each The Melanie II User Manual LABELS feature will be displayed with its own label You may display only one label tag for all identical labels by choosing Show Labels Pack Labels Show Labels Unpack Labels separates the label tags again Fig 6 1 Figure 6 1 A label for Human a bumin You can either display the label names or the label ACs The option Show Labels Show AC and Show Labels Show Name may be used to tog gle the AC name display You may show only selected labels with Show Labels Selected Labels Modifying and deleting labels In the Edit Labels sub menu two options allow you to modify and delete existing labels Edit Labels Modify Labels is available when one label is selected This pops up the label window You may then change the AC or name Edit Labels Delete Labels deletes all selected labels You can manually change a label s tag position Select a label with the Label tool Then move the cursor onto the label s tag Hold down the mouse button and drag the tag Release the mouse button The Melanie II User Manual 6 3 6 ES LEES Reports on labels Select labels using the Label tool or the Select Labels options Choose Show Labels Report On Labels A report will be displayed giving specific information on the selected labels s
2. 4 1 x golaris 1 or Solaris 2 and either X Windows X11R4 X11R5 or OpenWindowsa3 We recommend at least 100 MB of swap space to process several gels simultaneously Melanie I 2 D PAGE release 2 2 for Macintosh requires the following minimal hardware environment Apple Power Macintosh and 32 MB of memory We recommend 64 or even 128 MB of memory to pgess many gels simultaneously Alternatively you may use a Power Macintosh with less memory and define virtual memory though performances will not be as good Programs and files distributed with the Melanie Il 2 D PAGE package Melanie Il 2 D PAGE programs Mel1View Melanie II main program MelBatch program to perform actions in batch mode The Melanie II User Manual OVERVIEW MelLicense license manager Unix only MelPprogress progress report program Unix only elmadmin Melanie II license administrator Unix only elmd Melanie II license demon Unix only Public domain programs Melxwd Unix only Unix only Unix only Melgiftopnm Melpgmtops Unix only Melpgmtolpr Melpnmscale Unix only Unix only Unix only Unix only Melppmtogif Melppmtopgm Melxwdtoppm Master protein maps CSF mel Human cerebrospinal fluid ECOLI mel Escherichia coli ELC mel Human erythroleukemia cell HEPG2 mel HepG2 HEPG2SP mel HepG2 secreted proteins LIVER mel Human liver LYMPHOMA mel Human lymphoma PLASMA mel Human plasma PLATELET
3. Figure 4 4 The ID amp pl Mw window Additionally this option also displays the feature ID when the cursor is posi tioned over a detected feature see Features spots on page 5 1 4 4 The Melanie II User Manual er AE ad j FFF i _ FEATURES SPOTS The elementary component of a gel is the feature Features are spot shapes automatically detected by Melanie II s feature detection algorithm or manually adjusted by the user Each feature in a gel has an associated feature ID a unique sequential number automatically assigned to when it is created Fea tures may be quantified i e their optical density or instrument count for non optical images area and volume are computed Alternatively features may be modeled by Melanie s Gaussian modeling algorithm In this case their quantifi cation values will be the bi Gaussians peak values and volume as well the area of the Gaussian s half height section see Creating Gaussian gels on page 12 5 Detecting features Select gels to detect features on then take Process Detect Features The Detect Features window pops up Various detection parameters are available and may be adjusted The default parameters have been optimized for silver The Melanie II User Manual 5 1 5 2 5 HENNE FEATURES SPOTS stained 2 D PAGE images run according to the SWISS 2DPAGE protocol see reference 9 You may tune the parameters to adapt the detection program to your images The fol
4. see Aligning gels on page 7 1 then you may use the right mouse button to select labels in all aligned gels simultaneously See detailed discussion on labels in Labels on page 6 1 The Magnify tool The Magnify tool lets you zoom in on the region of a gel around the cursor posi tion Just pick the Magnify tool then hold down the mouse button Similar to a magnifying glass you may then move the mouse to change the zoomed region If gels have been aligned see Aligning gels on page 7 1 you may use the right mouse button to magnify the same region in all aligned gels simultaneously Menus Once objects have been selected you may choose actions from the menus to be performed on them Move the cursor to the desired menu on the menu bar located at the top of the MelView window hold down the mouse button then 1 On the Macintosh hold down the Command key 3 and the mouse button The Melanie II User Manual 2 9 2 NEE THE MELVIEW COMPONENTS 2 10 Iy PC Unix choose a menu option Certain menu options have sub options you may choose from by dragging the cursor over the small arrow on the right hand side of the option see Fig 2 6 S Melaniell Melview FA Fie EN Setect Show Stack Image Process Analyze Landmarks ET Add Feature Labels t Pairs Modify Features i Group t Delete Features Comments F ba wy Figure 2 6 Choosing a sub option from the menu The followin
5. 14 1 2 DE databases 14 1 Master gels 14 2 SWISS 2DPAGE master gels 14 2 Selecting CH2D features 14 4 Adding labels from a master 14 4 Updating labels from a master 14 4 Deleting labels from amaster 14 4 Querying a remote database through the Internet 14 5 PART IV THE MELBATCH PROGRAM 15 THE MELBATCH PROGRAM 15 1 Batch processing 15 1 Running MelBatch 15 1 Converting images with the Unix release 15 2 Reducing images 15 3 Detecting and quantifying features 15 3 Filtering gels 15 4 Matching and pasting landmarks 15 4 Matching gels 15 4 Creating synthetic gels 15 5 Printing images 15 5 Combining operations 15 6 Exiting MelBatch 15 6 PART V TUTORIAL 16 TUTORIAL 1 LOOKING AT GELS 16 1 Manipulating two gels 16 1 Opening two 2 DE images 16 2 Adjusting colors 16 5 IV The Melanie II User Manual CONTENTS Zooming 16 7 Stacking gels 16 7 Aligning gels 16 8 Magnifying glass 16 9 Exiting MelView 16 9 17 TUTORIAL 2 ANALYZING GELS 17 1 Analyzing a set of three gels 17 1 Setting preferences 17 2 Opening 2 DE images 17 3 Adjusting colors 17 6 Reporting on gels 17 7 Printing gels 17 7 Setting pseudo colors 17 8 Detecting and quantifying features 17 8 Reporting on features 17 10 Stacking gels 17 10 Aligning gels 17 10 Defining regions 17 12 Magnifying glass 17 12 Matching gels 17 13 Creating a synthetic gel 17 15 Matching gels to the synthetic gel 17 15 Grouping features 17 16 Using a master gel 17 17 Using the Internet
6. DE image that has been digitized and stored on disk in the Melanie Il 2 D PAGE release 2 format Images in other formats TIFF GIF PPM may also be opened as gels but they have to be saved into the Melanie II format in order to keep objects such as detected feature shapes feature val ues pairs etc Other file formats may only contain plain unstructured images If a gel is saved with its objects into a foreign format then the objects will simply be part of the saved image and will have lost any structure or value The gel image is the raw input data to the MelView program from which fea tures may be detected and quantified Gels may also be duplicated erased cropped filtered flipped or scaled Synthetic gels may be created by merging spots from other gels Regions A region is a rectangular subpart of a gel that can be defined using the Region tool When a region is defined certain actions may be limited to it Fig 2 4 Region Features Landmark Label Figure 2 4 Feature shapes a landmark and a label in a region Landmarks Individual points pixels in a gel may be marked by landmarks using the Landmark tool A landmark is defined by its position row and column number and its name It may be assigned p and MW values p and MW values of land marks are used to compute an approximated p and Mw value for any point in a The Melanie II User Manual 2 3 2 4 2 THE MELVIEW COMPONENTS gel Landmarks may also be u
7. Files column click Open Filter fhome antineargels_ecoli Directories a palagi gels_ecoli a palagi gels_ecoli afpalagi gels_ecoli Melanie afpalagi gels_ecoli pairs ECOLIT synthetic Fi Bi d a Ok Filter Cancel eal Figure 3 2 The Open window allows the selection of the gels that will be opened and loaded into MelView Unix release The Melanie II User Manual 3 3 3 4 3 ME Basic PROCEDURES Macintosh on each of the gel file names you want to open To choose discontinuous file names hold down the Shift Key and click on the file names Click on the OK button The gels will be opened The Open menu option allows you to open gel images stored on disk in the Melanie II release 2 or Melanie 1 format Melanie 1 images will be converted to Melanie II release 2 file format MelView will ask you for confirmation You can not open Melanie 1 images without converting them to the new iormat If you want to keep the image in the old Melanie 1 format use the Open As menu option see next section In Melanie Il for the Macintosh Click on the File menu and select the Open option The Open window will be displayed Fig 3 3 Click on each of the desired gel names and each time click Gels analyzed 94 0005 mel ECOLI mel 7 pairs Synthetic mel Open gels 94 0007 mel Figure 3 3 The Open window allows the selection of the gels that will be o
8. Landmarks to select all landmarks in all selected gels Finally if some landmarks have already been selected you have the possibility to do Select Landmarks Inverse Selection This will unselect the selected landmarks and select all unselected ones 1 On the PC or Macintosh you can use the sliders or the arrows 2 On the Macintosh hold down the Command key and the mouse button The Melanie II User Manual LANDMARKS Showing landmarks Landmarks may be displayed with the Show Landmarks Show Land marks menu option and hidden with the Show Landmarks Hide Land marks option You may show only selected landmarks with Show Landmarks Selected Landmarks Modifying and deleting landmarks In the Edit gt Landmarks sub menu two options allow you to modify and delete existing landmarks Edit Landmarks Modify Landmarks is available when one landmark is selected This pops up the landmark window You may then change the various parameters for the selected landmarks Edit Landmarks Delete Landmarks deletes all selected landmarks You can also manually change a landmark position Select a landmark with the Landmark tool Then move the cursor to the cross representing the landmark position Hold down the mouse button and drag the landmark Release the mouse button To interactively change a landmark s tag position select a landmark with the Landmark tool Then move the cursor onto th
9. MelView window Select File Print gt Print MelView Window The image of the whole MelView window is sent to the printer it means to the Postscript printer when using the Unix release Printing selected gels Select some gels using the Select tool Then choose Files Print Print Selected Gels A pop up window lets you choose the gel objects to be printed with the image such as Features Landmarks Labels Vectors Color Adjust ment Alignment amp Zoom The selected gels will be printed in full size 1 In the PC mouse with 2 buttons hold down the Control key and the right mouse but ton In the Macintosh hold down the Option key and the mouse button 2 On the Macintosh use instead The Melanie II User Manual BASIC PROCEDURES Saving gels Any changes carried out on gels have to be saved in order to be stored in the gel file Otherwise the changes will be lost when exiting the MelView program or when closing the gels Choose File Save Save Changes to save all changes performed on the selected gels or any of the individual saving options For example File Save Save Features will only save the detected feature shapes but not other changes If you save gels after having performed color adjustments the latter will be saved with the gel images After performing changes on gels you may revert to the saved status on disk by choosing one of the Revert options in menu File Current changes wi
10. Q The Melanie II User Manual THE MELVIEW COMPONENTS In the same way in Melanie II for the Macintosh for example you may activate Macintosh the Open Gels action by holding down the Command key 8 and the O key simultaneously lower or upper case This is indicated next to the Open menu option by the 3 0 sign Similarly you may exit MelView by hitting 0 The Melanie II User Manual 2 11 2 WENE THE MELVIEW COMPONENTS 2 12 The Melanie II User Manual ae fia i Unix PC Macintosh BASIC PROCEDURES This chapter presents basic MelView procedures such as opening saving and closing gels manipulating gel images such as changing colors and zoom modes and printing them On line help The manual is also available on line in the MelView program To consult it on line in Melanie II for Unix choose Preferences in the File menu Set the Melanie Home directory to the directory in which the man directory resides usually the same as the Melanie II programs in Melanie II for the PC or Macintosh make sure that the Help folder is located in the same folder as the MelView application Then in the Helo menu choose Tutorial On line user manual and index are available too To access the on line tutorial and user manual you must have the Netscape Navigator World Wide Web browser installed You may choose the table of content document from which you can read any of the on line manual chapters or you can r
11. image grey level is mapped to white and maximum grey level is mapped to black functions e g in order to highlight small light spots MelView lets you change the grey level mapping in two different manners You can define the minimum 0 and maximum grey levels 255 i e you may look at only the light or the dark regions of the images Fig 3 7 Or you can change The Melanie II User Manual 3 9 3 ME Basic PROCEDURES 3 10 the default linear mapping function to an exponential or logarithmic one Fig 3 8 and 3 9 screen image 255 black original 5D image min grey max grey level level Figure 3 7 Mapping the light image pixels grey levels 1016 to 3844 in the original image will appear black screen image 255 black original image 52 3844 min grey max grey level level Figure 3 8 Using a logarithmic mapping function light spots appear darker The Melanie II User Manual BASIC PROCEDURES Figure 3 9 Region of a gel showing the color adjustment from Figure 3 8 the left image shows the original image the right image shows the same gel area after mapping with a logarithmic function To change the mapping function select some gels then choose Select Select Mode Adjust Colors Then in the Adjust Colors window change the minimum and maximum grey levels as well as the mapping function You may increase or decrease the curvature of the exponential or logarithmic f
12. press the left mouse button then quickly release it In given situations you may also click the middle or the right mouse button Generally the right mouse button is used to manipulate corresponding objects on several visiblegels Unix The Melanie II User Manual 2 5 2 6 2 ENE THE MELVIEW COMPONENTS Macintosh PC Unix Macintosh Important The leit mouse button being the most freguently used we shall sometimes in this manual refer to it by just saying the mouse button or click on the mouse In Melanie II for the Macintosh the most common way to use the mouse is to move it so as to position the cursor over a given object then press the mouse button then quickly release it In given situations you may also hold down the Option key and click the mouse button at the same time or hold down the Command key 3 and click the mouse button Generally the Command key mouse button combination is used to manipulate corresponding objects on sev eral visible gels Dragging In Melanie Il for PC or Unix you may also push down the left mouse button and then without releasing it move the mouse in order to drag objects from one place to the other In given situations this is also possible using the middle or the right mouse buttons In Melanie II for the Macintosh you may also push down the mouse button and then without releasing it move the mouse in order to drag objects from one place to the other In given situ
13. the cursor to the bottom right position All features in the selected region will be selected and highlighted in green Features may also be selected from the Select gt Features sub menu Choose Select Features By ID and enter a feature ID to select the corresponding feature Choose Select Features By Master ID and enter a feature ID from the masier gel to select the corresponding features see Master gels on page 14 2 Choose Select Features By Group ID and enter a group ID to select the corresponding features see Groups on page 9 1 Choose Select Features Only Gaussians to only select features that have been modeled by a Gaussian function Select a region on a gel using the Region tool then Select Features In Region to select all features in the selected region or Select Features All Features to select all features in all selected gels If certain labels are selected you may also choose Select Features For Labels This will select all fea tures associated to currently selected labels see Labels on page 6 1 If you have already saved a Report on Features you may reselect all the fea tures by choosing Select Features From File A pop up window lets you choose the name of the file which contains the report Finally if some features have already been selected you have the possibility to do Select Features Inverse Selection This will unselect the selected fea ture
14. 17 18 Analyzing data 17 19 Exiting MelView 17 21 Other Melanie II features 17 21 PART VI APPENDIXES A ALGORITHMS A 1 Detecting and quantifying features A 1 Aligning A 3 Matching A 8 Creating synthetic gels A 10 Gaussian fitting A 12 Filtering gelimages A 17 B KNOWN BUGS B 1 C REFERENCES C 1 The Melanie project C 1 The Melanie II User Manual V EE CONTENTS Two dimensional electrophoresis 2 DE C 2 2 DE databases C 2 The ExPASy WWW server C 3 D TECHNICAL SUPPORT D 1 INDEX l 1 Credits vi The Melanie II User Manual BASICS Using this manual Welcome to Melanie Il 2 D PAGE analysis software Melanie II 2 D PAGE is a complete state of the art software to view process and analyze images of two dimensional electrophoresis 2 DE This manual is divided into six parts The first three parts are devoted to Mel View Melanie s main program Part gives an overview of the Melanie Il 2 D PAGE software as well as the basic procedures for analyzing 2 DE images Part Il details the necessary steps for comparing images Part IIl pre sents advanced features such as editing image creation and working with remote databases as well as analyzing resulting data Part IV is dedicated to MelBatch a companion program that provides 2 DE analysis in batch non interactive mode The two tutorials in part V describe typical sessions with the MelView software using 2 DE gels provided on the distribution CD ROM We stron
15. LE Lid 1004 103 Da TT E A J TS Leed Eend 9 O M NA LA 7 009 iL GEN LIN Pe EO i els anal voed Beal i ia DLE PE De d TIP OES GIL LAM Poe OH ike ala anal voed E OTA LAN MM j k 20S JEI MES iS MAR FLiSU Gabor reed End AL dma NIE LAE 148 1 H1 5 13 Ut Eo E land ar ana reed Eno My Om Dak Hd d i Oo Tal nia Sey 8 0 1110 Galan ee rod Eed EI DHE NEE LET 1a 1 5 11 WE MAE Ge 1a Gel aan road Sol Tl O M FIT LIE 18 Ed EIN WHIL AHARD id oe FEdri eee wed Fol ol OMIE BEL Hd N dd OER EA TEJ MHART FH MEN Figure 5 5 Report on features To have master gel information M S N and M AC on the report it must be a SWISS 2DPAGE gel and it must be selected as the master gel see Master gels on page 14 2 Selecting unselecting all The Select Select All menu option lets you select all defined objects from selected gels including landmarks features and labels The Select Unse lect All option lets you unselect all objects from selected gels The Melanie II User Manual 5 7 5 MES FEATURES SPOTS Showing hiding all The Show Show All menu option lets you display all defined objects from selected gels including landmarks features and labels The Show Hide All option lets you hide all objects from selected gels Hiding objects also has the side effect of displaying images faster 5 8 The Melanie II User Manual Bres ad de FFP F Ca Iy Iy LABELS The label is a small tag associated to a feature and i
16. MELANIE II 2 D PAGE release 2 2 User Manual Catalog Number PC Macintosh Unix 170 7634 170 7632 170 7566 For Technical Service Call Your Local Bio Rad Office or in the U S Call 1 800 4BIORAD 1 800 424 6723 P N 4000103 RevA Warranty Bio Rad Laboratories warrants that the Melanie II software shall substantially conform in all operational features to Bio Rad s current specifications as pub lished in Bio Rad s user and installation guides and that when properly installed it will be free of material defects which affect system performance The Purchaser must notify Bio Rad in writing within 30 days of delivery of the soft ware not including delivery of any subsequent modifications to the software of any defect If the software is found to be defective by Bio Rad Bio Rad s sole obligation under this warranty is to remedy the defect in a manner consistent with Bio Rad s regular business practices For a defect which adversely affects the performance of the software Bio Rad shall use its best efforts to cure such defect as soon as reasonably practicable after receipt of Purchaser s notice For minor defects Bio Rad shall use its best efforts to correct such minor defects in the next release of its software If however Bio Rad is unable to cure a major defect within 90 days of receipt of Purchaser s notice Purchaser shall have the option to cancel this agreement whereupon Bio Rad shall refund only the software fees
17. Note that if the small rectangle temporarily disappears from the display when the Adjust Colors window pops up slightly change one of the parameters Click on the OK button The grey levels or colors if you have set pseudo colors see next section will be adjusted in all selected gels In Melanie II for the Macintosh note that if you want to move the MelView win dow while the Adjust Colors window is displayed you have to hold down the Command key 38 and drag the window with the mouse Selecting pseudo colors Choose Select Select Mode Pseudo Colors Choose one of the color lookup tables for example FiveRamps Click OK The lookup table will be The Melanie II User Manual BASIC PROCEDURES changed accordingly Fig 3 11 To go back to grey levels choose Select gt Select Mode Grey Levels P rei kla es Edi skei 7 ote NES i z RR z e FiveRamps color lookup table EER EE Figure 3 11 Gels displayed using the Selecting grey levels This is the default mode To select this mode choose Select Select Mode Grey Levels Stacking gels In given situations it is helpful to stack two gels i e to display one gel on top of the other By switching from one gel to the other you may then better compare given gel areas Select two gels then Stack Stack Selected Gels The two gels will be displayed on top of each other To
18. ations this is also possible using the Com mand key 3 mouse button or Option key mouse button combina tions Note that when you want to move the MelView window while a dialog window is displayed you have to hold down the Option key and drag the window with the mouse Tools Objects on screen may be displayed manipulated or processed using the vari ous options from the MelView menus Prior to choosing an option from the The Melanie II User Manual THE MELVIEW COMPONENTS menu bar individual objects have to be selected using the tools provided in the left hand side tool bar Fig 2 5 z lt a Select tool ty lt Hand tool a Region tool Landmark tool _ a Feature tool _ Label tool s lt Magnify tool Figure 2 5 The MelView tool bar The Select tool The Select tool is used to select individual gels To select one gel pick the Select tool in the tool bar The Select tool will be surrounded by a small black rectangle showing that it is active Put the cursor in a gel and click on the mouse button The gel will be surrounded by a green line indicating that it has been selected To select more than one gel select the first one as described above then hold the shift key on the keyboard and select additional gels by clicking on them This rule holds for any kind of objects in MelView to select several objects of the same kind pick th
19. bjects may also be selected using one of the tools from the left hand side vertical tool bar or using certain menu options for exam ple Select Features All Features Once selected an object is marked in green The green color is always used to highlight selected objects If an object is shown i e if it is visible this does not mean it has been selected As a gen eral rule actions from the menus may only be performed on selected objects Objects may also be hidden so that they are not visible on screen Cr Mouse buttons The mouse may be used in various manners each having a specific behavior depending on the active tool See Tools on page 2 6 Mouse click In Melanie II for PC the most common way to use the mouse is to move it so as PC to position the cursor over a given object then press the left mouse button then quickly release it In given situations you may also click the middle or the right mouse button Generally the right mouse button is used to manipulate corre sponding objects on several visible gels With a 2 button mouse the middle button is replaced by pressing the Control key and the right button simulta neously Important The left mouse button being the most frequently used we shall sometimes in this manual refer to it by just saying the mouse button or click on the mouse In Melanie II for Unix the most common way to use the mouse is to move it so as to position the cursor over a given object then
20. dentifying the correspond ing protein The label holds the protein s accession number AC and name taken from a user selected database or it may just contain any textual data The label is the link to MelView s remote database query engine Note that the AC is the label s identification Two features labelled with the same AC are considered identical while the label names may differ Also as labels are associated to features you may define labels only after hav ing detected features see Features spots on page 5 1 Creating labels To create a label pick the Label tool and double click on a feature you wish to associate a label to The Label window pops up Fill in the AC accession num ber and name fields Click on OK The label is created and its AC or name is displayed on the gel depending on the Show Labels options previously cho sen Alternatively labels may be created with the Edit Labels Add Labels option Select one or more features using the Feature tool then choose Edit gt Labels Add Labels The label will be added to all selected features The Melanie II User Manual 6 1 6 2 6 ES LEES Selecting labels Labels may be selected with the Label tool Click on any label to select it The selected label will be highlighted in green To select more than one label select the first one by clicking on it with the mouse button then hold the shift key on the Keyboard and select additional labe
21. ditional gels by clicking on them Alternatively you may select all visible gels in the MelView window by choosing Select Gels Visible Gels or only the aligned gels by taking Select Gels Aligned Gels See Aligning gels on page 7 1 or just the stacked gels by taking Select Gels Stacked Gels See Stacking gels on page 3 13 Showing gels reporting on gels Once you have opened a given number of gel images you can decide which ones you want to display and which ones you prefer to hide at a given point in time The Gels option in menu Show provides the following choices to this pur pose Selected Gels displays all selected gels and hides the other ones Aligned Gels displays all aligned gels See Aligning gels on page 7 1 and hides the other ones Stacked Gels displays all stacked gels See Stacking gels on page 3 13 and hides the other ones All Gels displays all opened gels including the currently hidden ones Show Comments displays the comments for the selected gel See Editing showing comments on page 11 5 The Melanie II User Manual 3 5 3 6 3 ME Basic PROCEDURES Report On Gels Displays a report Summarizing information about the selected gels such as image size in pixels minimum and maximum grey levels number of detected features Fig 3 4 GalHama Foez Cole HEFaasarei Goeeyleweles Eee Harrer Fiwdid k Tyra Pee aaa 3 Pry Gelade ijai rel mud LDE Figure 3 4 Report
22. dy detected using option 1 and modeling is performed on the detected features Note that the parameters are of no utility here Click OK Features spots will be detected according to the parameter values you have set After detection the feature shapes will be displayed over the gels Fig 5 2 and 5 3 Try first to detect features using the default parameter val ues then tune the parameters for your data og oc Oo 0 zi a Oy o 0 so SS oO ii oc amp Pa DE Figure 5 3 Features modeled as Gaussians When using 16 bit images a pop up window lets you choose before deciding the detection parameters wheter you want to change or not the grey levels to correspond to 12 bit images The Melanie II User Manual 5 3 5 ME FEATURES SPOTS CF od detection parameters in real Each change of a parameter value may immediately be reflected in a given region if such a region has been defined Select the Region tool and draw a small rectangle in one of the selected gels using the button mouse In the Pro cess menu choose Detect Features Each time you change one of the parame ters the change will immediately be reflected in the selected region Fig 5 4 EE L L ad Zi Desi ring Bed ed kpe Laie a al M Dest He HE sd aak EER OF emrlerg karre rand Deeps oe wa Ed sd gt x si 2 Figure 5 4 Adjusting detection parameters in real time Note that if the small rectangle tempo
23. e corresponding tool from the tool bar select the first object by clicking on it with the mouse button then hold the shift key on the key board and select additional objects by clicking on them The Hand tool The MelView window is often not large enough to display the whole gel images and only a small part of the gels will be visible at one given time The Hand tool moves gels in order to show other parts Pick the Hand tool in the tool bar put the cursor in a gel hold down the mouse button and drag the mouse The gel image will follow your movement If instead you hold down the middle mouse button then all selected gels will move together 1 On the PC mouse with 2 buttons hold down the Control key and the right mouse but ton On the Macintosh hold down the Option key and the mouse button The Melanie II User Manual 2 7 2 8 2 ENE THE MELVIEW COMPONENTS By holding down the right mouse button you will position all gels according to the absolute cursor position in the pointed gel That is the top left corner of each visible gel area will correspond to the same pixel in absolute row and col umn positions The Region tool The Region tool is useful to select a rectangular subregion of a gel Position the cursor at the top left position of the desired region hold down the mouse but ton then drag the cursor to the bottom right position A rectangle will be drawn around the region while selecting it Release t
24. e feature select the first one by clicking on it with the mouse button then hold the shift key on the keyboard and select additional features by clicking on them To select all features in a given region position the cursor at the top left position of the desired region hold down the mouse button then drag the cursor to the bottom right position All features in the selected region will be selected and highlighted in green The selected region delimits an area of the gel which given actions will be limited to 1 On the Macintosh hold down the Command key 3 and the mouse button The Melanie II User Manual THE MELVIEW COMPONENTS If gels have been aligned see Aligning gels on page 7 1 you may use the right mouse button to select features in all aligned gels simultaneously See detailed discussion on features in Features spots on page 5 1 The Label tool With the Label tool you may select labels To select a label pick the Label tool and click on the chosen label To select more than one label select the first one by clicking on it with the mouse button then hold the shift key on the keyboard and select additional labels by clicking on them To select all labels in a given region position the cursor at the top left position of the desired region hold down the mouse button then drag the cursor to the bottom right position All labels in the selected region will be selected and highlighted in green If gels have been aligned
25. e landmark s tag Hold down the mouse button and drag the tag Release the mouse button Fig 4 2 Figure 4 2 Default landmark tag position left and modified tag position right The Melanie II User Manual 4 3 A ES ANOMARKS Reports on landmarks Select landmarks using the Landmark tool or the Select Landmarks options Choose Show Landmarks Report On Landmarks A report will be dis played giving specific information on the selected landmarks such as landmark names positions and pl Mw values The report may be saved or printed Fig 4 3 T E a i il d des i 1E JISE TE 214500 TE 24000 AE 11A ME de lj ele Beis Pi 0 Tels Em1 Fars 00 Be Taio 7 lm Fran Fi 1 aa Ha 11AEE aad Ed Sele Em Pawn 7 ON Bed LIE di I Ei Bou fa L imi mi bei oi ei bei i weld Held il meld l we Figure 4 3 Report on landmarks A dialog box lets you choose the directory and the file name to save the report to Showing boland Mw F howing pl and Mw If you have defined a few landmarks in a gel and given pl and Mw values you can display the interpolated p and Mw values at any point in the gel Take Show ID amp pl Mw This will display a small window holding the pl and Mw at cursor position Moving the cursor will change the window content accordingly Fig 4 4 Gel Name belesEcoli Feature ID 1013 Row 454 Column 230 Pixel value 1E Izoelectric point 5 19 Molecular weight 46589
26. ed gels 7 2 Unaligning gels 7 4 Special alignments 7 4 MATCHING GELS 8 1 il The Melanie II User Manual CONTENTS Introduction 8 1 Matching aligned or unaligned gels 8 2 Automatic matching 8 2 Creating starting pairs 8 3 Using landmarks as starting pairs 8 4 Using labels as starting pairs 8 4 Showing pairs 8 4 Selecting pairs 8 5 Reports on pairs 8 6 PART Il ADVANCED FEATURES 10 11 12 GROUPS 9 1 Introduction 9 1 Selecting groups 9 2 Reports on groups 9 2 Histograms on groups 9 2 Groups with synthetic gels 9 3 EXPORTING DATA 10 1 Exporting to Outgroups 10 1 Exporting to ASCII tables 10 2 Exporting to ASCII lists 10 2 EDITING 11 1 Editing landmarks 11 1 Editing features 11 2 Editing labels 11 4 Editing pairs 11 5 Editing groups 11 5 Editing showing comments 11 5 CREATING IMAGES 12 1 The image menu 12 1 Duplicating gels 12 1 Erasing gels 12 2 Cropping gels 12 2 Filtering gels 12 2 Creating synthetic gels 12 4 Creating Gaussian gels 12 5 Flipping gels 12 6 Scaling gels 12 6 Rotating gels 12 6 Inverting grey levels 12 7 The Melanie II User Manual DS CONTENTS 13 DATA ANALYSIS 13 1 Introduction 13 1 Reports and histograms on groups 13 1 Specifying classes 13 2 Differential analysis with classes set 13 2 Student T Test with classes set 13 3 Differential analysis without classes set 13 4 Correspondence analysis without classes set 13 4 Reports on analysis 13 5 14 DATABASES
27. elView icon You may also run the MelView program by double clicking on the icon of a gel image file In this way the given gel will automatically be opened upon running the program Alternatively you may select several gel image files and choose File Open in the Macintosh menu or even drag and drop gel icons onto the MelView icon Note that if you have transferred images to the Macintosh using a program such as Fetch this option is only available if you specified MELA as the creator and TIFF as the type during file transfer The Melanie II User Manual OVERVIEW Upon loading the program the Melanie II 2 D PAGE logo appears Click on the logo to get rid of it The Melanie II User Manual 1 9 1 NEE OVERVIEW 1 10 The Melanie Il User Manual i j Ab r A Fa d on EEN A ETS E ER i ae n y r d n f r i i i a oe i M fie d d ia di ag r w CaP i Ta E Py Pas TA fs or a Je x F k EEH i if T MK Fr Pep FF k T hs i i O Mi PO N N l lt a Tp Ea aE roer ery m M n 4 Fa ah ee iF ii i l Poe z T sir og Fa Pie oe e dd Fa ia EF fii a s er F The MelView window is divided into three parts the horizontal menu bar on the top the vertical tool bar on the left side of the window and the rectangular dis play area Figs 2 1 2 2 and 2 3 show the starting MelView display on PC Macintosh and Unix respectively In the remaining part of this manual figures i
28. engine Fig 2 4 Pairs and groups Gels may be compared using Melanie s powerful automatic gel matching pro gram When two gels are matched the corresponding features are paired Selected feature pairs may be displayed when gels are in tile mode Alterna tively all pairs may be shown as small vectors when two gels are displayed in stack mode Gels can be matched pair wise or a set of gel images can be matched to a user chosen reference gel A reference gel may be any 2 DE gel image a master gel image or even a synthetic gel image The pair is the link between corresponding features i e the same protein in several gels When several gels have been matched to a given reference gel the latter pro vides a unique feature numbering scheme across all gels Each paired feature in a gel image may then be associated to the corresponding feature ID in the reference gel The features in a set of gels that are paired to one given feature in the reference gel form a feature group The group is the basic element for exporting data as tables and for producing histograms of feature values across a series of gel images The Melanie II User Manual THE MELVIEW COMPONENTS Comments Textual data may be associated to individual gels in the form of comments Comments may be displayed printed or saved to files Lr Selecting and showing objects Objects may be either shown hidden or selected When an object is shown it is visible on the screen O
29. equest the on line index then click on the desired concept to obtain the related on line chapter You may also directly refer to the on line tutorial Go to the Help menu and choose one of the avail able options The Melanie II User Manual 3 1 3 2 3 ME Basic PROCEDURES Unix PC Macintosh PC If Netscape was running before MelView and if the colors in Netscape do not look correct quit Netscape and choose the Help option again Setting preferences A few preferences may be set and preserved between MelView sessions In menu File choose Preferences You may set the following preferences In Melanie II for Unix 1 The Netscape Navigator full pathname To use the on line manual as well as the database options see Databases on page 14 1 you must give the exact location of the Netscape program for example usr local bin netscape 2 The database server To use the database options see Databases on page 14 1 you must specify the hostname of the World Wide Web server on which the database resides for example www expasy ch 3 The database name To use the database options see Databases on page 14 1 you must specify the remote database for example SWISS 2DPAGE 4 The printer You have to give the printer name for example ID 5 The Melanie Home directory This is the directory in which the on line manual files resides the man directory usually the same as the Melanie II programs In Melanie II for PC o
30. g menus are provided in the MelView program from left to right in the menu bar 1 File to handle whole gels and data on disk open and close gel images save data print images export data 2 Edit to edit add modify delete selected objects 3 Select to select specific objects 4 Show to show or hide specific objects as well as to display reports 5 Stack to handle stacked gels two gels on top of each other 6 Image to create new gel images from selected ones duplicate crop filter gels create synthetic or Gaussian gels flip and scale gels as well as erase gels 7 Process to detect and quantify features and to align and match gels 8 Analyze to compute differences and similarities among gels statistical data analysis 9 Database to handle master gels and to access remote 2 DE databases 10Help to obtain on line help At given points in time depending of the status of the MelView window certain menu options will not be available In this case they will appear faded in the menus Shortcuts Several menu options may be activated by keyboard shortcuts These are indi cated at the right hand side of the corresponding menu option In Melanie II for PC or Unix for example you may activate the Open Gels action by holding down the Control key and the O key simultaneously lower or upper case This is indicated next to the Open menu option by the o sign Similarly you may exit MelView by hitting
31. gly suggest you read through this section before working with Melanie II 2 D PAGE Part VI provides technical support information Finally an index is supplied at the end of the manual Appendix A details the algorithms and methods used in Melanie II 2 D PAGE Important points in the manual as well as useful hints are marked by a small hand in the margin in order to draw your attention The manual is also available on line in the MelView program See On line help on page 3 1 The Melanie ll User Manual 1 1 1 2 1 NEE OVER VIEW Iy PC Unix Macintosh Important note This manual describes the Melanie Il 2 D PAGE software release 2 2 for PC Macintosh and Unix The functionality and usage are similar on the three ver sions Where necessary we highlight the differences Also files are placed in directories that are called folders on the Macintosh For sake of clarity we always refer to directories Macintosh users will rectify easily Hardware and software requirements Environment Melanie I 2 D PAGE release 2 2 for PC compatible requires the following mini mal hardware environment Windows 95 or MS Windows NT 4 0 or later and 32 MB of memory We rec ommend 64 or even 128 MB of memory to process many gels simultaneously Melanie Il 2 D PAGE release 2 2 for Unix requires the following minimal hard ware and software environment Sun Miggosystems workstations SPARCstation series 32 MB of memory SunOS
32. he mouse button at the desired point You may move the region by dragging the rectangle s top left corner You may also change its size by dragging its bottom right corner If gels have been aligned see Aligning gels on page 7 1 then you may use the right mouse button to select the same region in all aligned gels simultaneously The Landmark tool The Landmark tool is used to create or select landmarks at specific points on the gel image To create a landmark pick the Landmark tool then double click the mouse button at the position you want the landmark to be To select an existing landmark just click on it To select more than one landmark select the first one by clicking on it with the mouse button then hold the shift key on the keyboard and select additional landmarks by clicking on them To select all landmarks in a given region position the cursor at the top left position of the desired region hold down the mouse button then drag the cursor to the bottom right position All landmarks in the selected region will be selected and high lighted in green If gels have been aligned see Aligning gels on page 7 1 you may use the right mouse button to select landmarks in all aligned gels simultaneously See detailed discussion on landmarks in Landmarks on page 4 1 The Feature Tool The Feature tool lets you select individual features To select a feature pick the Feature tool and click on the chosen feature To select more than on
33. he reference gel may then be clustered to form groups A group represents the same spot over a set of gels Reports on groups as well as his tograms may be displayed printed or saved Groups may also be exported to ASCII tables in order to be used with external programs such as Excel or other data analysis packages Analyzing sets of gels Statistical data analysis may be performed on groups of features in sets of gels Variations differences and similarities are computed Classes of gels may be created to represent different populations Reports on the analysis results as well as graphics may be displayed printed or saved Creating new images Gel images may be duplicated flipped scaled or cropped Filters are provided to improve image quality such as contrast enhancement smoothing and back ground subtraction Synthetic gels may be produced by merging spot informa tion from several gels Gaussian modeling of features is also supplied Querying databases Master gels may be defined or those provided on the distribution CD ROM may be used A master gel contains labels for each identified protein These proteins may then be automatically linked to remote databases such as SWISS 2DPAGE or SWISS PROT through the use of Netscape Navigator MelBatch In the MelView program actions may be carried out on open gels only The MelBatch program is supplied to perform certain actions on gels without the need of displaying them In this wa
34. hen enter the following sequence of commands gt cd pub navigator Right Version unix gt bin dir Find the correct version corresponding to your computer and operating system SunOs or Solaris then type gt get Right Version KOULE where Right Version is the version you want to download Uncompress the downloaded file and follow the installation instructions To download the Macintosh version of this software use Fetch a freeware program or any other FTP program Double click on the fetch icon Enter the following information Host FTP site where FTP site is one of the FTP sites above User ID anonymous The Melanie II User Manual 1 5 1 6 1 NEE OVER VIEW Password your full electronic mail address Directory pub navigator Right_version mac Click OK Then select the latest version of Netscape and click on Get Files Provide a file name to save the program and click OK Once the program is downloaded double click on its icon Overview of Melanie II 2 D PAGE Melanie Il 2 D PAGE is a complete state of the art software application designed to analyze images of two dimensional electrophoresis 2 DE You may perform qualitative and quantitative image analysis of 2 DE gels MelView is the main program and provides interactive as well as automatic gel analysis Manipulating images 2 DE images may be manipulated in various ways Images may be displayed in tile mode one gel next to the other or in stack
35. hole image Non optical images The pixel value is instrument counts Count the highest instrument count in the feature AREA the feature s area VOL the feature s volume i e the integration of Count over the feature s area Count the relative Count i e the feature Count divided by the total Count over the whole image VOL the relative VOL i e the feature VOL divided by the total VOL over the whole image Gaussian features lf features have been modeled as Gaussians the following quantification values are computed OD Count the optical density respectively count of the Gaussian s peak AREA the area of a section of the Gaussian taken at the peak s half height VOL the Gaussian s volume OD the relative OD i e the OD divided by the total OD over the whole image The Melanie II User Manual 5 5 5 6 5 ME FEATURES SPOTS VOL the relative VOL i e the VOL divided by the total VOL over the whole image Selecting features Features may be selected with the Features tool Click on any feature to select it The selected feature will be highlighted in green To select more than one feature select the first one by clicking on it with the mouse button then hold the shift key on the keyboard and select additional features by clicking on them To select all features in a given region position the cursor at the top left position of the desired region hold down the mouse button then drag
36. in a gel image may be marked by a landmark A land mark is defined by its position row and column number and its name It may be assigned p and Mw values p and Mw values of landmarks are used to compute approximated p and Mw value for any point in a gel Landmarks may also be used as reference points for operations such as gel alignment and gel matching see Aligning gels on page 7 1 and Matching gels on page 8 1 Creating landmarks To create a landmark pick the Landmark tool and double click at the position in a gel where you want the landmark to be located The Landmark window pops up Enter a name You may also enter the p and or Mw of the gel at the given position A value of 1 means that no value is set The pop up windows also indicates the selected landmark position in row and column numbers You can adjust these by clicking on one of the row or column sliders and moving them hold down the mouse button and drag the cursor To assure fine position adjustment click on one of the sliders then adjust the position using the arrow keys Click on OK The landmark is created and its name and position are dis played on the gel The Melanie II User Manual 4 1 4 2 A EE ANOMARKS Iy If the landmark contains a pl or Mw value then its tag will contain an asterisk Fig 4 1 Figure 4 1 A landmark with pl Mw shown by the asterisk Alternatively landmarks may be created with the Edit Landmarks Add Landmark
37. l then hold down the mouse button Similar to a magnifying glass you may then move the mouse to change the zoomed region Fig 3 5 Figure 3 5 The magnifying glass If gels have been aligned see Aligning gels on page 7 1 then you may use the right mouse button to magnify the same region in all aligned gels simulta neously Changing colors A 2 DE gel image is traditionally displayed as a grey level image where grey levels represent the gels optical density respectively instrument counts for non optical images You may opt to display images using pseudo colors You may also change the way grey levels are displayed Adjusting colors Modern scanners are usually able to scan 2 DE images with 12 or even 16 bits per pixel that is with 4096 or 65536 grey levels respectively Because common screens are only able to display 256 colors or grey levels mapping has to be undertaken between the 4096 or 65536 grey levels respectively and the 256 screen grey levels By default MelView uses a linear mapping function where the lightest point in the image is mapped to O white and the darkest point is 1 On the Macintosh hold down the Command key 3 and the mouse button The Melanie II User Manual BASIC PROCEDURES mapped to 255 black Fig 3 6 However you may choose other mapping screen image 255 black original image 52 3844 min grey max grey level level Figure 3 6 default linear mapping minimum
38. ll be lost If objects have never been saved to disk for the selected gels then the Revert option is not active Alternatively you may save any gel images in one of the supported foreign file formats In this case you can choose to save the gel image alone or the gel image with any of its associated components such as features labels or land marks But the saved file will contain a flat image without any structure For example when saving a gel with its features to a TIFF file this file will contain a flat image of the gel and its features Choose File Save As then select the objects you want to add to the image and click OK The File Extension window will pop up You can then enter the names of the new files manually or use the Replace Extension or the Add Extension options Add Extension lets you spec ify an extension to add to each new file name while Replace Extension lets you replace the file name extensions of the selected gels with any other extension Closing gels and exiting MelView Use File Close to close all selected gels If any of the gels have not been saved since changes were performed MelView lets you choose to save them before closing them To exit MelView do File Quit MelView lets you choose to save any unsaved gel images before exiting The Melanie II User Manual 3 15 3 MEN Basic PROCEDURES 3 16 The Melanie II User Manual Pree 7 Frea dd LANDMARKS Individual points pixels
39. llustrate screens from the PC version only The display is similar on Unix and Macintosh When necessary screen displays from specific platforms will be shown BID RAD Kl LLE Melanie ll Een mae ic ee a Thi ln kan bh ik mg kuier ur med nl Rig Pere Figure 2 1 The MelView display on the PC The Melanie II User Manual 2 1 2 NEE THE MELVIEW COMPONENTS Melanie ll Figure 2 2 The MelView display on the Macintosh Amal ele rs Eile dai ri Re Em imr Erase pulpe Foie Hele Be Pet e Leg BIO RAD Melanie ll Cary gry lee Ter Pet SS Sie ee ee 1 Ad Sis Eara ed Figure 2 3 The MelView display on Unix You may use the tools to select objects in the display area then apply actions from the various menu options to the selected objects Objects are gel images landmarks associated to specific points on the image features detected spots labels associated to features feature pairs between two gels groups of features across several gels or comments on gels This chapter describes the functionality of the various MelView components Their practical use will be discussed in detail in the following chapters 2 2 The Melanie II User Manual THE MELVIEW COMPONENTS Objects A Melanie Il object is an entity that may be displayed and manipulated The objects are gels as well as gel components such as regions landmarks fea tures labels pairs groups or comments Gels A gel is a 2
40. lowing parameters may be set Fig 5 1 Detect Features Select detection parameters Number of smooths E E 2 Laplacian threshold HE 3 Partials threshold aj gt 1 Saturation al E oe Feakedness increase ET 100 Minimum perimeter Mm mi gt 10 Detect using default algorithm Laplacian feature shapes Detect then fit with Gaussians Fit existing features with Gaussians Cancel Default Figure 5 1 The feature detection parameters Number of smooths on the images Before detecting features MelView will smooth the image using a smooth by diffusion algorithm in order to eliminate parasite noise Select the number of times the smoothing operation should occur Note that other smoothing algorithms are available in the mage menu see Filtering gels on page 12 2 To avoid smoothing set the parameter to zero Threshold for Laplacian The detection algorithm looks for spot peaks by cal culating the Laplacian for each pixel in the image To detect as many features as possible set a small value for this parameter If you set high values only very curved features will be detected and flat spots will be ignored Threshold for Partial derivatives Melanie also checks the spot curvature in both the X and Y axes in order to separate features with flat sections or to eliminate streaks Augmenting this value increases the separation power of the detection algorithm OD resp counts above which image i
41. ls by clicking on them To select all labels in a given region position the cursor at the top leit position of the desired region hold down the mouse button then drag the cursor to the bottom right position All labels in the selected region will be selected and highlighted in green Labels may also be selected from the Select Labels sub menu Choose Select Labels By AC and enter a label accession number to select the corresponding labels You may use wildcards to select a group of labels For example P12 designates all labels whose ACs start with the letters PAZ Choose Select Labels gt By Name and enter a label name to select the cor responding labels You may also use wildcards to select a group of labels Select a region on a gel using the Region tool then Select Labels In Region to select all labels in the selected region or Select Labels AI Labels to select all labels in all selected gels If you have selected features before selecting labels you may take Select gt Labels For Features and all the labels associated to the selected features will be selected Finally if some labels have already been selected you have the possibility to do Select Labels Inverse Selection This will unselect the selected labels and select all unselected ones Showing labels Labels may be displayed with the Show Labels Show Labels menu option and hidden with the Show Labels
42. mel Human platelet RBC mel Human red blood cell U937 mel Macrophage like cell line YEAST mel Yeast Saccharomyces cerevisiae Example images Three images in directory Gels Same images after complete analysis in directory Gels analyzed Six Plasma gels after complete analysis in directory Gels analyzed Other files Manual and tutorial files in directory man Unix or Help PC or Macintosh Licence hardware protection key for PC version To run MelView you need to install the provided hardware protection key on the parallel port see the Release Notes The Melanie II User Manual 1 3 1 4 1 ES OVERVIEW Licence hardware protection key for the Macintosh version To run MelView you need to install the provided hardware protection key between the computer and the keyboard see the Release Notes Netscape Navigator In order to use the on line manual as well as to access remote databases over Internet you must install the Netscape Navigator World Wide Web browser from Netscape Communications Corp Mountain View California USA You can purchase this product from Netscape Communications Corp or download a free copy for non commercial use by anonymous FTP from the following FTP sites that are available at Netscape Communications Corp FIP sites ftp netscape com ftp1 netscape com fip2 netscape com fip3 netscape com fip4 netscape com ftpo netscape com fip6 netscape com fip7 netscape com Director
43. mode two gels on top of each other They may be zoomed Various color modes are provided in order to best visualize spots Selected 2 DE images or the whole MelView screen may be printed Images may be imported from or exported to various file formats such as TIFF GIF and PPM Detecting and quantifying features Melanie Il automatically detects features spots on several gels at once Vari ous reports on feature values such as optical density or instrument counts for non optical images area and volume are computed and may be displayed printed or saved to files Landmarks and labels You may add landmarks to gels in order to mark specific points on the image Landmarks may just hold a name or they can be associated to p and Mw val ues They may also be used as starting points for the gel matching program Labels may be defined and associated to detected features They are most often used to mark identified proteins They hold the accession numbers iden tification numbers and names of the proteins Aligning and matching gels Gels may be aligned that is distorted gels will be stretched pixel wise in order to make a set of images completely superimposable Aligned or non aligned The Melanie II User Manual OVERVIEW gels may be automatically matched i e corresponding features will be paired across gels Grouping features A set of gels may be matched to one reference gel All features paired with the same spot in t
44. on gels Report On Matches Displays information on matching between the selected gels and all open gels that is the number of paired features between each pair of gels See Matching gels on page 8 1 Report All Matches Displays information on matching between all gels in a given directory See Matching gels on page 8 1 A pop up window requests the directory name default current directory Moving gels The size of the MelView window usually does not allow to display whole gels At any point in time you may determine to move gel images in order to show other image parts You may also change the way in which gels are arranged on the screen Showing corners The Show Corner option lets you choose to display the top left top right bot tom left or bottom right part of the selected gels Just take Show Show Cor ner then choose the corresponding sub option Using the Hand tool The Hand tool allows you to interactively move one or several gels Pick the Hand tool in the tool bar put the cursor in a gel hold down the mouse button and move the mouse The gel image will follow your movement The Melanie II User Manual BASIC PROCEDURES If instead you hold down the middle mouse button then all selected gels will move together By pressing the right mouse button you will position all gels according to the absolute cursor position in the pointed gel That is the top left corner of each visible gel area will corresp
45. ond to the same pixel in absolute raw and column position If you cannot select a gel because it was moved outside the visible parts of the screen while using the hand tool then select all gels with Select Gels gt Visible Gels and Show Show Corner Using the Select tool You may change the order in which gel images are arranged on the screen Pick the Select tool put the cursor in the gel you want to shift then hold down the mouse button and move the cursor over another gel Release the mouse button The two gels will be swapped Zooming Another method to decide what part of a gel should be displayed is to change the zoom mode You may zoom in to enlarge the image you will see less of the image at once or zoom out to see more of the gel image Selecting Zoom mode Select the gels you want to zoom then take Select Select Zoom and choose the zoom mode 1 is the default mode and displays images in real size 2 zooms in by a factor of 2 1 2 and 1 4 zoom out by a factor of respectively 2 and 4 1 On the PC mouse with 2 buttons hold down the Control key and the right mouse but ton On the Macintosh hold down the Option key and the mouse button 2 On the Macintosh hold down the Command key d and the mouse button The Melanie II User Manual 3 7 3 8 3 ME Basic PROCEDURES Using the magnifying glass An alternative way to enlarge parts of gels is to use the magnifying glass Pick the Magnify too
46. paid The warranties set forth in this agreement are in lieu of all other representations and warranties expressed or implied including war ranties of merchantability and fitness for a particular purpose and any other statutory or common law warranty Bio Rad on its own behalf expressly dis claims and excludes any and all such other representations and warranties Liability of Bio Rad to Purchaser if any for breach of warranty or any other claim relating to this agreement shall be limited to the total amount of software fees paid by purchaser to Bio Rad In no event shall Bio Rad be liable for inci dental or consequential damages loss of business or profits special or indirect damages of any nature whatsoever No amendment waiver or other alteration of the warranties in this agreement may be made except by mutual agreement IN writing Purchaser agrees that Bio Rad s liability arising out of contract negligence Strict liability in tort or warranty shall not exceed the amount of software license fees paid by Purchaser Under the copyright laws this manual and the software program contained herein may not be copied in whole or in part without the prior written consent of Bio Rad except in the normal use of the software or to make a backup copy This exception does not allow copies to be made for oth ers whether or not sold but all of the materials purchased with all backup cop les may be sold given or loaned to another person Unde
47. pened and loaded into MelView Macintosh release on the Add button or just double click on each gel name This adds the requested gels to the Open gel box Click on the Done button The gels will be opened Opening foreign image formats MelView allows you to import images stored in foreign image file formats such as TIFF 8 or 16 bits GIF or PPM In the Unix release MelView also lets you import the Elsie and Melanie 1 formats Choose File Open As In the Open As window that pops up choose one of the available formats Click OK In the next pop up window select the images you want to open following the above described procedure Click on the OK button on the Done button on the Macin tosh Opening a Melanie II image in this way lets you open a copy of the gel image The Melanie II User Manual BASIC PROCEDURES Selecting gels To perform the actions described in the remaining part of this chapter you must first select one or more gels This may be achieved using either the Select tool or one of the Gels options in menu Select To select one gel pick the Select tool in the tool bar The Select tool will be sur rounded by a small black rectangle showing that it is active Put the cursor ina gel and click on the mouse button The gel will be surrounded by a green line indicating that is has been selected To select more than one gel select the first one as described above then hold the shift key on the keyboard and select ad
48. r Macintosh 1 The database server To use the database options see Databases on page 14 1 you must specify the hostname of the World Wide Web server on which the database resides for example www expasy ch 2 The database name To use the database options see Databases on page 14 1 you must specify the remote database for example SWISS 2DPAGE In the Preferences window click on the OK button If preferences were already found on disk MelView will ask you to confirm the new preferences Opening gels Opening Melanie 2 DE images In Melanie II for PC The Melanie II User Manual Unix BASIC PROCEDURES Click in the File menu and select the Open option The Open window will be displayed Fig 3 1 Hold down the Shift key and in the Files column click on Open gels EE Path CAM elarniell Gels analpzed 94 0002 mel Chercher Gels analzed Eg E synthetic mel Nene 34 0002 mel Type Gel images mel 7 Annuler Selected 34 0002 mel Figure 3 1 The Open window allows the selection of the gels that will be opened and loaded into MelView PC release each of the gel file names you want to open To choose discontinuous file names hold down the Control Key and click on the file names Click on the OK button The gels will be opened In Melanie II for Unix Click on the File menu and select the Open option The Open window will be displayed Fig 3 2 Hold down the Control key and in the
49. r the law copying includes translating into another language or format A multi use license may be purchased to allow the software to be used on more than one computer owned by the purchaser including a shared disk system Copyright Copyright 1985 1997 The Melanie Group Geneva Switzerland All rights reserved Copyright 1991 Free Software Foundation Inc for the GNU C library Copyright 1990 1993 Groupe BULL for the XPM library Copyright 1994 1995 Quest Protein Database Center Cold Spring Harbor Labs for the gd library Copyright 1988 1996 Sam Leffler and 1991 1996 Silicon Graphics Inc for the TIFF library Trademarks TIFF is a trademark of Aldus Corporation Windows 95 and Windows NT are trademarks of Microsoft Corporation Sun Microsystems SPARCSta tion OpenWindows and SunOs are trademarks or registered trademarks of Sun Microsystems Inc Apple Macintosh and Power Macintosh are trademarks of Apple Computer Inc UNIX is a trademark of Novell Inc PostScript is a trademark of Adobe Systems Inc Netscape Communica tions Netscape and Netscape Navigator are trademarks of Netscape Communications Corporation FrameMaker is a registered trademark of Frame Technology Corporation Rather than put a trademark symbol in every occurrence of other trade marked names we state that we are using the names only in an editorial fashion and to the benefit of the trademark owner with no intention of infringement of
50. rarily disappears from the display when the Detect Features window pops up change slightly one of the detection parameters Click on the OK button The features will be detected in all selected gels using the parameter values you have set Quantifying features Choose Process Quantify Features to compute the feature values Depend ing on the scanner used to digitize the images the following quantification val ues are calculated for each detected feature 5 4 The Melanie II User Manual FEATURES SPOTS Images digitized by transparence The pixel value is the optical density OD the highest optical density in the feature AREA the feature s area VOL the feature s volume i e the integration of OD over the feature s area OD the relative OD i e the feature OD divided by the total OD over the whole image VOL the relative VOL i e the feature VOL divided by the total VOL over the whole image Images digitized by transmittance The pixel value is transmittance A conversion table is used during quantifica tion in order to convert transmittance values to optical density OD the highest optical density in the feature AREA the feature s area VOL the feature s volume i e the integration of OD over the feature s area OD the relative OD i e the feature OD divided by the total OD over the whole image VOL the relative VOL i e the feature VOL divided by the total VOL over the w
51. s and select all unselected ones Showing features Features may be displayed with the Show Features Show Features Shapes menu option and hidden with the Show Features Hide Features Shapes option You may show only selected features with Show Features Selected Features The Melanie II User Manual Iy LF FEATURES SPOTS You may also show respectively hide the IDs of selected features Select some features then Show Features gt Show Features ID respectively Show Features Hide Features ID Hiding features also has the side effect of displaying images faster Reports on features Select features using the Feature tool or the Select gt Features options Choose Show Features Report On Features A report will be displayed giving specific information on the selected features such as features ID quanti fication values pl and Mw as well as the following data from the master gel provided the selected gels have been matched to a master gel see Matching gels on page 8 1 and Master gels on page 14 2 serial number pl Mw and accession number M S N M pl M Mw and M AC respectively The report may be saved or printed Fig 5 5 Report in feiren vd Aard road Ecel ML GEI HEN Dk NA OUE did MAA HA 1MH Ged ase voed Eai Ai OES DEE DIE dl OUE EE LIN THARA EIS JE ed ee voed EO Ab DOME HALE Dl LAF OB LE eS EL 11 Ged anaeal reed Beal i ai hi Ed DA
52. s option In this case the Landmark window pops up without prior landmark position You then have to specify the landmark position using the sliders or the arrow keys For aligned gels see Aligning gels on page 7 1 double click using the right mouse button in order to create the same landmark at the same position in all selected aligned gels Selecting landmarks Landmarks may be selected wit the Landmark tool Click on any landmark to select it The selected landmark will be highlighted in green To select more than one landmark select the first one by clicking on it with the mouse button then hold the shift key on the keyboard and select additional landmarks by clicking on them To select all landmarks in a given region position the cursor at the top left position of the desired region hold down the mouse button then drag the cursor to the bottom right position All landmarks in the selected region will be selected and highlighted in green Landmarks may also be selected from the Select Landmarks sub menu Choose Select Landmarks By Name and enter a landmark name to select the corresponding landmark You may use wildcards to select a group of landmarks For example L designates all landmarks whose names start with the letter L Select a region on a gel using the Region tool then Select gt Landmarks In Region to select all landmarks in the selected region or Select Landmarks All
53. s saturated in percentage Some very dark spots may be saturated especially when digitized with older scan ners and may therefore be flat on the top To insure that these features be detected Melanie II artificially increases the peakedness curvature of all pix els whose OD respectively counts are above a given threshold The threshold is expressed as a percentage of the maximum grey level in the image A value of 90 is usually considered correct Increase in peakedness for saturated pixels This is an internal value expressing how much Melanie increases the curvature of saturated pixels 100 is the default value and should only be changed with care The Melanie II User Manual FEATURES SPOTS Minimum feature perimeter With this parameter you may decide to ignore features that are too small Melanie II will ignore all features whose perimeters in pixels are smaller than the given threshold An additional criterion affects the way features are represented You may choose between the following three options Detect using default algorithm This is the default Features are detected using the Laplacian and feature shapes are represented that match the original spot shapes Detect then fit with Gaussians Features are detected with the Laplacian then Gaussian modeling is performed on all detected features Features are represented by bi Gaussian functions Fit existing features with Gaussians Features have to be alrea
54. sed as reference points for operations such as gel alignment and gel matching Fig 2 4 Features The elementary component of a gel is the feature Features are spot shapes automatically detected by Melanie II s feature detection algorithm or manually adjusted by the user Fig 2 4 Gels may be displayed with or without their fea ture shapes Each feature in a gel has an associated feature ID a unique sequential number automatically given when it is created Features may be quantified i e their optical density or instrument count for non optical images area and volume are computed Alternatively features may be modeled by Mel anie s Gaussian modeling algorithm In this case their quantification values will be the bi Gaussians peak values and volume as well as the area of the Gauss lans half height section Features represent the proteins from the original image Features may auto matically be paired between two gels or groups of features may be formed across a series of gels Quantification values of spot groups may then be dis played or printed as histograms or exported as tables for use with external data analysis programs Labels The label is a small tag associated to a feature and identifying the correspond ing protein The label holds the protein s accession number AC and name taken from a user selected database or it may just contain any textual data The label is the link to MelView s remote database query
55. switch from one gel to the other choose Stack Front To Back or just Control F By pressing Control F quickly several times the differences between the two gels can be observed visually To unstack the gels choose Stack Unstack Selected Gels 1 On the Macintosh use 3 instead The Melanie II User Manual 3 13 3 ME Basic PROCEDURES 3 14 Moving stacked gels MelView provides a means to superimpose corresponding features of stacked gels in a simple way Select the Hand tool When two gels are stacked and selected you may use the mouse button to move the front gel By holding down the middle mouse button both gels will be moved at the same time To superimpose two features put the cursor on the given feature in the front gel then hold down the mouse button Press Control F to display the other gel Move the cursor to the corresponding feature and release the mouse but ton The two features will be superimposed Verify this by pressing Control F Printing gels Two print modes are provided in MelView You may print selected gels one gel image per page or print the whole MelView window In Melanie II for Unix you first have to set the printer Select Files Prefer ences Make sure to define an existing printer example lp You may also set the printer from the File Print Printer Setup option In Melanie II for the PC or Macintosh select File Page setup and verify the settings Printing the
56. the trademark Production notes This document was created electronically using FrameMaker 5 PARTI BASICS Warranty OVERVIEW 1 1 Using this manual 1 1 Important note 1 2 Hardware and software requirements 1 2 Overview of Melanie Il 2 D PAGE 1 6 The Melanie Il 2 D PAGE file format 1 8 Running MelView 1 8 THE MELVIEW COMPONENTS 2 1 Objects 2 3 Selecting and showing objects 2 5 Mouse buttons 2 5 Tools 2 6 Menus 2 9 BASIC PROCEDURES 3 1 On line help 3 1 Setting preferences 3 2 Opening gels 3 2 Selecting gels 3 5 The Melanie II User Manual EE CONTENTS Showing gels reporting on gels 3 5 Moving gels 3 6 Zooming 3 7 Changing colors 3 8 Stacking gels 3 13 Printing gels 3 14 Saving gels 3 15 Closing gels and exiting MelView 3 15 4 LANDMARKS 4 1 Creating landmarks 4 1 Selecting landmarks 4 2 Showing landmarks 4 3 Modifying and deleting landmarks 4 3 Reports on landmarks 4 4 Showing pl and Mw 4 4 5 FEATURES SPOTS 5 1 Detecting features 5 1 Adjusting detection parameters in real time 5 4 Quantifying features 5 4 Selecting features 5 6 Showing features 5 6 Reports on features 5 7 Selecting unselecting all 5 7 Showing hiding all 5 8 6 LABELS 6 1 Creating labels 6 1 Showing labels 6 2 Modifying and deleting labels 6 3 Reports on labels 6 4 PART II ALIGNING AND MATCHING 7 ALIGNING GELS 7 1 Introduction 7 1 Creating corresponding landmarks 7 2 Aligning gels 7 2 Operations on align
57. uch as label AC and name as well as fea ture ID and position The report may be saved or printed Fig 6 2 PR HH Jit g th ite an Li Mr o Bart Figure 6 2 Report on labels 6 4 The Melanie II User Manual
58. unction by adjusting the respective sliders Fig 3 10 Hold down the left mouse button and move the slider Click OK Colors will be adjusted in all selected gels If you save gels after having performed color adjustments the latter will be saved with the gel images see Saving gels on page 3 15 Adjusting colors in real time Each change of the mapping function in the Adjust Colors window may immedi ately be reflected in a given region if such a region has been defined prior to adjusting colors Select the Region tool and draw a small rectangle in one of the selected gels by putting the cursor at the top left point of the region holding down the mouse button and dragging the cursor to the bottom right point Release the mouse button The selected region will be delimited by a rectangle In the Select menu choose Select Mode Adjust Colors Each time you 1 On Unix you can also click once on the slider and adjust it using the arrow keys On the Macintosh you can also adjust the slider using the arrows The Melanie II User Manual 3 11 3 ME Basic PROCEDURES 3 12 Macintosh change one of the mapping parameters the function or a slider the change will immediately be reflected in the selected region Fig 3 10 bin paar suk ae KIN ajj ajo Him seel ales EN aj T Linn T fupra E Dore al al H de a Figure 3 10 The Adjust Colors option The modifications are directly reflected in the selected region
59. y pub navigator Right version windows for the PC version pub navigator Right_version unix for the Unix version pub navigator Right_version mac for the Macintosh version where Right Version is the version you want to download Mirror sites exist in other geographical areas and you may download Netscape from them To obtain an updated list of FTP sites get the following document from Netscape Communications Corp s Web site http home netscape com comprod netscape_products html or Netscape Communications Corp s main Web page http home netscape com To download the PC version of this software type the following command gt ftp EIRP Ssate The Melanie II User Manual OVERVIEW where FTP site is one of the FTP sites above Enter anonymous as user name and give your full electronic mail address as password Then enter the following sequence of commands gt cd pub navigator Right Version windows ar bani gt dir Find the correct version corresponding to your computer and operating system 16 or 32 bits then type gt get Right Version KOULE where Right Version is the version you want to download Double click on the downloaded file and follow the installation instructions To download the Unix version of this software type the following command gt ftp FTP site where FTP site is one of the FTP sites above Enter anonymous as user name and give your full electronic mail address as password T
60. y feature detection and quantification fol lowed by gel matching may be performed on a set of gels as a background pro cess The Melanie II User Manual 1 7 1 8 1 NEE OVER VIEW PC Unix Macintosh The Melanie II 2 D PAGE file format The Melanie Il programs work with Melanie II release 2 file formats as well as with TIFF files Analyzed gels are always saved into the Melanie II release 2 file format unless otherwise specified by the user Gel images may be imported from or exported to other formats such as TIFF GIF and PPM Running MelView To run the MelView program PC release double click on the MelView icon You may also run the MelView program by double clicking on the icon of a gel image file In this way the given gel will automatically be opened upon running the program To run the MelView program Unix release type the following command gt MelView amp The amp sign indicates that the MelView program has to run in background You have to make sure that the directory in which the MelView program resides is part of your path Type gt echo Spach to verify this If it is not in the path please refer to the Unix manual to include it You may also run the MelView program and automatically open gel images Type the MelView command followed by the gel names For example to open gels GelA and GelB type gt MelView GelA GelB amp To run the MelView program Macintosh release double click on the M

Overview of Melanie II 2-D PAGE - Bio-Rad

Contents

Download Pdf Manuals

Related Search

Related Contents