TUTORIAL - Bio-Rad
Contents
1. 1 On the Macintosh use instead 2 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 3 On the Macintosh hold down the Command key 3 and the mouse button The Melanie II User Manual 17 11 17 TUTORIAL 2 ANALYZING GELS Defining regions e Select the Region tool third tool in the tool bar In the ECOLI gel hold down the mouse button and drag the mouse for about one inch Release the button A small rectangle will be drawn defining a region on the ECOLI gel Now hold down the right mouse button drag the mouse Release the mouse button The same region will be defined in all gels Magnifying glass e Select the Magnify tool last tool in the tool bar Click the mouse button and move the cursor in any of the gels A small area under the cursor will be magni fied Now hold down the right mouse button and move the cursor The same region will be magnified in all aligned gels at the same time Fig 17 12 fo Melaniell Melview Me Ea HA File Edt Select Show Stack Image Process Analyze DataBase Help la x Figure 17 12 The Magnify tool applied to aligned gels 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 17 12 The Melanie II User Manual TUTORIAL 2 ANALYZING GELS Matching gels We wi2. Click OK The L1 landmark will be created on the second gel Repeat this step four times in order to add four landmarks around each of the four corners of the gels Press Control R to unstack the gels Press Control S to save the gels Now that both gels contain related landmarks we are going to perform the align ment Choose Process Align Gels Align Gels In the Align Gels window select gel 94 0002 as the reference gel Click OK Gel 94 0005 will then be aligned relative to 94 0002 Choose Stack Stack Selected Gels to stack the gels again To visually com pare both gels switch from one gel to the other by pressing Control F quickly several times 1 On the Macintosh use instead 2 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 TUTORIAL 1 LOOKING AT GELS Magnifying glass e Press Control R to unstack the gels Select the Magnify tool last tool in the tool bar Click the mouse button and move the cursor in one of the gels A small area under the cursor will be magnified Now hold down the right mouse button and move the cursor The same region in both gels will be magnified at the same time Fig 16 9 eg Melaniell Melview Iof x HA File Edt Select Show Stack Image Process Analize DataBase Help l x Figure 16 9 The Magnify tool applied to aligned gels
3. Exiting MelView To exit MelView do File Quit To try out more advanced features such as detecting spots and matching gels you may proceed to Tutorial 2 analyzing gels on page 17 1 1 On the Macintosh use instead 2 On the Macintosh hold down the Command key amp and the mouse button The Melanie II User Manual 16 9 16 ES TUTORIAL 1 LOOKING AT GELS 16 10 The Melanie IlI User Manual PC Macintosh Unix TUTORIAL 2 4 ANALYZING GELS Introduction This section describes a typical session with the Melanie Il software using the gels provided on the distribution tape in directory Gels You may read this section and at the same time perform the described steps The directory Gels analyzed contains the same gels after all the analysis described in this tuto rial has been performed You may therefore check what the result of this analy sis should be To do the complete analysis perform the following steps The gt sign indicates the command prompt on the Sun workstation Analyzing a set of three gels In Melanie II for PC or Macintosh Go to the directory Gels in which the sample gels are located Double click on the MelView icon In Melanie II for Unix Go to directory Gels in which the sample gels are located for example usr local MelanielII Gels 1 On the Macintosh a directory is called a folder The Melanie II User Manual 17 1 17 TUTORIAL 2 ANALYZING GELS g
4. 002 gels Choose Show Gels Selected Gels then Stack Stack Selected Gels The two gels are now displayed on top of each other To 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 Aligning gels This step is optional but it is very useful when analyzing gels that are not com pletely superimposable Gel alignment corrects gels pixelwise according to a reference gel in order to make all gels superimposable 1 On the Macintosh use instead The Melanie II User Manual TUTORIAL 2 ANALYZING GELS The first step consists on setting a few landmarks in each of the three gels Each landmark must correspond to the same feature in all gels Start with the two currently stacked gels The landmarks must be well distributed over the gel Follow the next steps Put the ECOLI gel in front Select the Landmark tool fourth tool in the tool bar Press Control F to toggle To add a landmark double click in the center of the ECOLI gel on a spot that is also in the other two gels The Create Landmark window pops up Enter a landmark name for example L1 Click OK The landmark now appears on the gel image Fig 17 11 Figure 17 11 The L3 Landmark Press Control F to toggle to gel 94 0002 To move the top gel so that a spot is exactly above the corresponding spot in the other gel follow t
5. 2 94 0623 and 94 0624 in the Files column This selects the six gels Click on the OK button The gels will be opened In Melanie II for the Macintosh Click in the File menu and select the Open option The Open window will be displayed Select the Gels Analyzed directory by double clicking in it Click on each of the six gel files called 94 0613 94 0614 94 0616 94 0622 94 0623 and 94 0624 and each time click on the Add button This selects the six gels and adds them to the Open gel box Click on the Done button The gels will be opened All six gels have already been analyzed Gel 94 0623 was used as the refer ence map In the Select menu choose the Gels Visible Gels option This will select all gels and draw a green line around each of them showing that they have been selected In MelView the green color always indicates selected objects In the Show menu choose Hide All to hide the already detected and annotated fea tures in the gels Select the Hand tool the second tool in the tool bar Place the cursor in one of the gels Hold down the right mouse button and move the gels until you see a region with many spots 1 On the Macintosh hold down the Command key 3 and the mouse button The Melanie II User Manual 17 19 17 17 20 TUTORIAL 2 ANALYZING GELS Differential analysis with classes set To use this analysis option you need to create two classes of gels To create the first class pick the Select tool
6. II logo to get rid of it Opening two 2 DE images pc In Melanie II for PC 16 2 The Melanie II User Manual TUTORIAL 1 LOOKING AT GELS e Click on the File menu and select the Open option The Open window will be displayed Fig 16 2 Hold down the Control key and click on gels 94 0002 and ee eee ee ee nR Fah E Hass h aarep HE ow Dechy acire a Goll a eE l i Ta Sal raga ral if 7 Salcied 44 Tie Figure 16 2 The Open window allows the selection of the gels that will be opened and loaded into MelView PC release 94 0005 in the Files column This selects the two gels Click on the OK button The gels will be opened Unix In Melanie II for Unix Click on the File menu and select the Open option The Open window will be displayed Fig 16 3 Hold down the Control key and click on gels 94 0002 and Open Filter fhomeantineagels_ecoli Directories ECOLI synthetic J p Pe J _ _ OK Filter Cancel Figure 16 3 The Open window allows the selection of the gels that will be opened and loaded into MelView Unix release The Melanie II User Manual 16 3 16 TUTORIAL 1 LOOKING AT GELS 16 4 Macintosh 94 0005 in the Files column This selects the two gels Click on the OK button The gels will be opened In Melanie Il for the Macintosh e Click on the File menu and select the Open option The Open window will be displayed Fig 16 4 Click on gel
7. OLI gel is a SWISS 2DPAGE master gel This means that several pro teins have been identified on the 2 DE map Corresponding entries exist in the SWISS 2DPAGE database on the Expasy World Wide Web server Each iden tified protein has a label associated with the corresponding feature e Choose Database Set Master Select ECOLI The ECOLI gel is now defined as the master gel Its name is printed with a red background e Select ECOLI with the Select tool then Control Z to have it alone on the screen e Choose Show Labels Show Labels All existing labels will appear show ing the SWISS 2DPAGE accession numbers ACs of all identified proteins Fig 17 18 Figure 17 18 Labels showing the proteins accession numbers 1 On the Macintosh use instead The Melanie Il User Manual 17 17 17 TUTORIAL 2 ANALYZING GELS e Choose Show Labels Show Name The SWISS 2DPAGE short protein names will be shown instead of the accession numbers Fig 17 19 Figure 17 19 Labels showing the proteins short names ID e Choose Show Gels All Gels to display all gels again then Control B then Select Features All Features Finally choose Database Labels Add From Master Labels will be propagated to paired features If necessary choose Show Labels Show Labels Fig 17 20 Figure 17 20 Labels propagated to other gels Using the Internet lf your computer is connected
8. PARI V TUTORIAL PC Macintosh Unix TUTORIAL 1 LOOKING AT GELS Introduction This section describes a simple session with the Melanie Il software using the gels provided on the distribution tape in the directory Gels You may read this section and at the same time perform the described steps The gt sign indicates the command prompt on the Sun workstation This tutorial explains how to open gels then how to manipulate them in order to improve their visualization Tutorial 2 analyzing gels on page 17 1 presents more advanced features of the Melanie II software Manipulating two gels In Melanie II for PC or Macintosh Go to the directory Gels in which the sample gels are located Double click on the MelView icon In Melanie II for Unix Go to directory Gels in which the sample gels are located for example usr local MelanielII Gels 1 On the Macintosh a directory is called a folder The Melanie II User Manual 16 1 16 TUTORIAL 1 LOOKING AT GELS gt cd usr local Melaniell Gels e Type the following command to run the MelView program gt MelView amp e The amp sign indicates that the MelView program has to run in the background Figure 16 1 shows the screen that appears when running MelView m 1F Bet ee bees bees Dee bide BIO RAD Melaniell ea fi Ce gee ic ia T Th Mia ee bh ap eer Ee pe a Age BELE AH Figure 16 1 The MelView main screen e Click on the Melanie
9. Select a logarithmic color func tion and adjust its parameter to the value 4 The change will directly be reflected in the selected region Fig 16 6 cg Melaniell Melview Me F File Edit Select Show Stack Image Process Analyze DataBase Help x Select Color Adjustment Parameters Min pixel value 0 4 Manx pixel value 2355 al Linear Exponential re rl 4 Cancel Default Figure 16 6 The Adjust Colors option The modifications are directly reflected in the selected region e Click on the OK button The colors will be adjusted in the two selected gels 16 6 The Melanie II User Manual TUTORIAL 1 LOOKING AT GELS Zooming e With the Select tool active first tool in the tool bar select only one gel Then in the Show menu choose Gels Selected Gels Only the selected gel stays in the MelView window The other gel is hidden In menu Select choose Select Zoom 1 2 The gel is zoomed out by a factor of 2 and a larger part of the gel can be seen Fig 16 7 So Melaniell Melfiew OF x HA File Edt Select Show Stack Image Frocess Analyze DataBase Help _ o x Figure 16 7 A gel after it has been zoomed out Stacking gels e Choose Show Gels All Gels and Select Gels Visible Gels then Stack gt Stack Selected Gels The two gels are now displayed on top of each other To switch from one gel to the other choose Stack Front To Back or just Con trol F By pressin
10. al 17 13 17 TUTORIAL 2 ANALYZING GELS e A second way of viewing results of the matching operation is to select a few fea tures in the reference gel ECOLI using the Feature tool then to choose Select Pairs For Features This will select and display the paired features in the other two gels Fig 17 13 ej Melaniell Melview Miel EA File Edit Select Show Stack Image Process Analyze DataBase Help _ o x Figure 17 13 Selected paired features across the three gels e Thirdly you may select two gels then select a few features on them then Show Pairs Report On Pairs to get the feature IDs of the pairs e Finally select gel 94 0005 and ECOLI then Control Z and Control T The two selected gels are now stacked Choose Show Pairs Show Pairs All the paired features will be shown by small blue vectors Fig 17 14 a ha L s aen an gt at i ete ro Oy Figure 17 14 Matched gels blue vectors indicate paired features 1 On the Macintosh use instead 17 14 The Melanie IlI User Manual TUTORIAL 2 ANALYZING GELS Creating a synthetic gel Let us now create a synthetic gel by merging the three gels Prior to the cre ation of a synthetic gel pairwise matching has to be performed on all gels involved Therefore we still have to match 94 0002 with 94 0005 Choose Show Gels All Gels then select gels 94 0002 and 94 0005 Choose Process Match Match Landmarks then Pr
11. e analysis without classes set Keep the same region as in the preceding paragraph and select groups in this region choose Select Groups In Region Choose Analyze Without Classes Set Correspondence Analysis Select the value type for example the feature area Accept the default value of the number of significant groups that will be dis played in the results See Correspondence analysis without classes set on page 13 4 To get a report on correspondence analysis results choose Show Analysis Report On Analysis You may print or save the report You can also visualize the results by displaying a projection of gels and features in the 2 D factorial space Choose Show Analysis gt Graphics on Analysis Choose to display gels and significant features Exiting MelView To exit MelView do File Quit Other Melanie II features Many other options and features are available in the MelView program includ ing Importing exporting images from and to TIFF GIF PPM etc Filters for smoothing contrast enhancement background subtraction etc Managing gel images flipping scaling duplicating etc Landmark feature and label selection by various criteria Landmark feature label pair and group editing The Melanie II User Manual 17 21 T TUTORIAL 2 ANALYZING GELS 17 22 The Melanie IlI User Manual
12. g Control F quickly several times the differences between the two gels can be observed visually 1 On the Macintosh use instead The Melanie II User Manual 16 7 16 16 8 TUTORIAL 1 LOOKING AT GELS Aligning gels We are now going to align the two gels in order to correct their relative distor tion The first step consists in setting a few landmarks in the two gels Each landmark must correspond to the same feature in both gels The landmarks must be well distributed over the gel Follow the next steps Put gel 94 0002 in front Select the Landmark tool fourth tool in the tool bar Press Control F to toggle To add a landmark double click in the center of the gel on a spot that is also in the other gel The Create Landmark window pops up Enter a landmark name for example L1 Click OK The landmark now appears on the gel image Fig 16 8 Figure 16 8 The L3 Landmark Press Control F to toggle to gel 94 0005 To move the top gel so that a spot is exactly above the corresponding spot in the other gel follow these steps with the Landmark tool selected put the cursor in the center of a feature spot Hold down the middle mouse button and at the same time press Control F once and move the cursor to the center of the corresponding spot in the other gel Release the mouse button Double click on the same feature spot in gel 94 0005 The same landmark name should already be shown in the Create Landmark window
13. ges All pairs will be preserved Process Align Gels gt Unalign Gels Grouping features When several gels have been matched with one reference gel the paired fea tures form so called groups A group is a set of features all paired together Figure 17 16 shows three such groups Groups may be visualized in reports and in histograms They may also be exported to files in order to be used in external programs such as Excel To get a report on groups choose Show Groups Report On Groups Select a gel as reference gel for example ECOLI If a master gel has already been set the latter will be taken as reference gel See Using a master gel on page 17 17 Select the value type for example the optical density OD or the feature area 1 On the Macintosh use instead The Melanie II User Manual TUTORIAL 2 ANALYZING GELS e To get histograms on groups choose Show Groups Histograms On Groups Figure 17 17 shows histograms for two of the selected features The bels analyzed 94 0002 a bels analyzed 94 0005 b Gels analyzed ECOL E Gels analyzed Synthetic d abcd abed Group 1002 Group 1017 Figure 17 17 Histograms for two feature groups IDs of the selected groups are shown on the reference gel e To export the groups choose File Export Select the export format You may export to Outgroups format this is the Melanie 1 table format Excel tables and Excel lists Using a master gel The EC
14. hese steps with the Landmark tool selected put the cursor in the center of a feature spot Hold down the middle mouse button and at the same time press Control F once and move the cursor to the center of the corresponding spot in the other gel Release the mouse button Double click on the same feature spot in gel 94 0002 The same landmark name should already be shown in the Create Landmark window Click OK The L1 landmark will be created on the second gel Repeat this step four times in order to add four landmarks near each of the four corners of the gels Press Control R to unstack the gels Choose Show Gels All Gels Then with the Select tool select the ECOLI and 94 0005 gels Press Control Z and Control T to display the two selected gels in stack mode Add the same five landmarks to gel 94 0005 Choose Show gt Gels All Gels then Select Gels Visible Gels Press Control S to save the gels Now that all three gels contain related landmarks we are going to perform the alignment Choose Process Align Gels Align Gels In the Align Gels win dow select the ECOLI gel as the reference gel Click OK Gels will then be aligned relative to the ECOLI gel Once gels have been aligned many tools and actions that work on one gel also work on all gels at the same time Instead of pressing the mouse button to use a tool on one gel press the right mouse button The tool will be applied to all aligned gels
15. ications are directly reflected in the selected region e Click on the OK button The colors will be adjusted in all three selected gels 1 On the Macintosh hold down the Command key 3 and the mouse button 17 6 The Melanie II User Manual Unix PC Macintosh TUTORIAL 2 ANALYZING GELS Reporting on gels Choose Show Gels Report On Gels A window pops up giving basic information about the selected gels such as image size in pixels minimum and maximum grey levels number of detected features spots etc At this point only the ECOLI gel has detected features Fig 17 7 Feport Oo Gels Fore Cole UbhFewtures Grevieveie Aieorey Lecter Pigtsesh Tape Sele spelyped EOTLE eel Pe A 0 mai f f Figure 17 7 The Report On Gels window Printing gels You first have to set the printer In Melanie II for Unix select File gt Preferences Make sure to define an exist ing printer example Ip In Melanie II for the PC or Macintosh select File gt Page Setup and verify the settings Then 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 Using the Select tool now select two gels To achieve this click on one gel then hold down the shift key and click on another gel Choose File Print gt Print Selected Gels In the pop up window select Color Adjustment and c
16. in the tool bar Put the cursor in gel 94 0613 and click on the mouse button Then hold the shift key on the keyboard and select gels 94 0614 and 94 0616 by clicking on them This will select the three gels and draw a green line around each of them showing that they have been selected Choose Analyze Set Class A pop up window asks you to give a name to this class Accept the example name class A by clicking OK Now select gels 94 0622 94 0623 and 94 0624 as described above Choose Analyze Set Class and accept the example name class B by click ing OK Now select the Region tool the third tool in the tool bar and draw a small rect angle in one of the gels using the mouse and the button To select groups in this region choose Select Groups In Region Select gel 94 0623 as reference gel by choosing Database Set Master and pick ing up 94 0623 as reference gel Choose Analyze With Classes Set Differential Analysis Select the value type for example the feature area Accept the default ratio value 200 to be considered between the mean value of one class for a given group and the mean value for the other class on the same group See Differential analysis with classes set on page 13 2 If there aren t any features selected increase the rectangle size in one of the gels To get a report on differential analysis results choose Show Analysis gt Report On Analysis You may print or save the
17. lick on the OK button The selected gels will be printed in full size The Melanie II User Manual 17 7 17 TUTORIAL 2 ANALYZING GELS 17 8 Setting pseudo colors e Choose Select Select Mode Pseudo Colors Choose one of the color lookup table for example FiveRamps Click OK The look up table will be changed accordingly Fig 17 8 Choose Select Select Mode Grey Lev els to go back to normal grey level display r4 bhkta fh I ii e Figure 17 8 The gels displayed using the FiveRamps color lookup table Detecting and quantifying features e Select the gels that have not yet been analyzed 94 0002 and 94 0005 using the Select tool and holding down the shift key Choose Process Detect Fea tures Click OK Features spots will be detected using the default parameters The Melanie II User Manual TUTORIAL 2 ANALYZING GELS Now choose Process Quantify Features to compute the optical density area and volume of the detected features Fig 17 9 Select File Save gt Save Changes to save the features and their associated quantification values o Melaniell MelView Miel x File Edt Select Show Stack Image BEE Detect Features Detect DNA Features Align Gels Match ir os 0 a a am E aoe Bow a C L lt Ed n oT oa E Pe a E E E _ E a Figure 17 9 Features have been detected The Quantify Features opti
18. ll now compare the features spots in gels 94 0002 and 94 0005 with those in the ECOLI gel This process is called gel matching It is going to pair each feature in each of the two gels with the corresponding features in ECOLI The matching algorithm in Melanie II is able to automatically match features without any manual intervention Nevertheless when gels are quite dissimilar or just to speed up the matching process you may manually define starting fea ture pairs As we have already defined landmarks in all three gels we will use these as starting pairs Select all three gels using the Select tool or Control B Choose Process Match Match Landmarks This will perform pairwise matches on the features containing landmarks in our case landmarks L1 to L5 Choose Process gt Match Match Gels The Match Gels window pops up asking for the reference gel i e the gel the other selected gels will be matched with Choose ECOLI and click OK This will now compare all features in gels 94 0002 and 94 0005 to the features in gel ECOLI Depending on the type of computer you are using this might take a few minutes After matching has been successfully completed press Control S to save the pairs There are various ways of looking at the result First choose Show gt Gels gt Report On Matches A window shows how many features have been paired across the gels 1 On the Macintosh use instead The Melanie Il User Manu
19. ocess Match gt Match Gels Wait for the matching to finish Press Control S to save the pairs Choose Image Create Gels Create Synthetic Gels Then choose ECOLI as the reference gel This means that the new synthetic gel will be based on ECOLI adding spots from the other gels In the next pop up window give a name for the synthetic gel We will just call it Synthetic Once created the synthetic gel will be displayed You might display it in the full screen to have a better look at it select the gel then Control Z See Figure 17 15 Figure 17 15 A part of the synthetic gel displayed with GEColor Matching gels to the synthetic gel We are now going to use the synthetic gel as a reference gel because it con tains most of the important features from the three other gels Add the L1 to L5 landmarks to the synthetic gel See Aligning gels on page 17 10 1 On the Macintosh use instead The Melanie Il User Manual 17 15 17 TUTORIAL 2 ANALYZING GELS 17 16 e Match the three gels with Synthetic Control B then Process gt Match Match Landmarks Choose Synthetic as the reference gel Then Process gt Match Match Gels and choose Synthetic as reference gel again The three gels should now be matched with Synthetic Fig 17 16 eG wal el gt Pa Figure 17 16 Three selected pairs Once the gels have been matched you may unalign the gels in order to work with the original ima
20. on has been selected The Melanie II User Manual 17 9 17 TUTORIAL 2 ANALYZING GELS 17 10 Reporting on features e With the Feature tool fifth on the tool bar select a few spots click on one spot lt will be displayed in green showing that it has been selected Then hold down the shift key and click on a few other spots Choose Show Features gt Report On Features This brings up a report window showing specific informa tion on the selected features Fig 17 10 Haga Ce nar LE Report In feiere TL Ge pare load Rn l MLO ED OL EL OE dTi MA fe H oI ea ans ed Eli ae GEA LIE L3 p137 GIS EJE UAI eee 51E IEIS es Led ElL me GHE Ble Fil Oa Bd Wish HARS pT JEJA iela EL ar D7 pIE OL 1 053 1 03 6 2 LNI HT ED OES lym EL aa 0A KI LAO i03 iLa Sai MAR DA E jR lal EL aa DAA BE BES TTF OD SI W AT E is ireal E SO iea LA Gi jae piia 5J JEJ M N 8 OR Aid a pe pa a Wi owa iig Lak 143 iHi ni uhii Mee i E Guarana Lael a 1 Hr Od iim iim iia Oe Til ube Sey fon ini Cael cna lyd ea i WI OME Le OL 1 387 oe 5 1 LS Se OT JEN aan aad ci 1 I D E LID LJE J i 1 E I LES MAEN pao JEH Pea E e eed Bod HL O lt HE BL Nam 8 22 DEA ERD TR DET fa OB oan i r Figure 17 10 Report on selected features Stacking gels Choose Select gt Gels Visible Gels then Show Hide All to hide the detected features This also has the side effect of displaying the images faster Select the ECOLI and 94 0
21. on of the gels that will be opened and loaded into MelView PC release file names in the Files column This selects the three gels Click on the OK but ton The gels will be opened Unix In Melanie Il for Unix The Melanie II User Manual 17 3 17 TUTORIAL 2 ANALYZING GELS e Click in the File menu and select the Open option The Open window will be displayed Fig 17 3 Hold down the Control key and click on each of the three Open Filter fhome antineargels_ecoli Directories ar palagirgels_ecoli ecolirHelaniez Ta Figure 17 3 The Open window allows the selection of the gels that will be opened and loaded into MelView Unix release file names in the Files column This selects the three gels Click on the OK but ton The gels will be opened Macintosh In Melanie Il for the Macintosh 17 4 The Melanie II User Manual TUTORIAL 2 ANALYZING GELS e Click in the File menu and select the Open option The Open window will be displayed Fig 17 4 Click on each of the three gels and each time click on the Gels analyzed Y Macintosh HD Papel 94 0005 mel ECOLI mel C pairs Synthetic mel Open gels 94 0007 mel Figure 17 4 The Open window allows the selection of the gels that will be opened and loaded into MelView Macintosh release Add button This selects the three gels and adds them to the Open gel box Click on the Done button The gels will be opened e In
22. report Student T Test with classes set To visualize the Student T Test results keep the classes set classes A and B and select all groups in the six visible gels Choose Select gt Groups All Groups Choose Analyze With Classes Set gt Student T Test Select the value type for example the feature area Accept the default value of the T Test 0 95 to be considered between both classes See Student T Test with classes set on page 13 3 To get a report on Student T Test analysis results choose Show Analysis gt Report On Analysis You may print or save the report Differential analysis without classes set For this analysis method you have to unset the existing classes Choose Analyze Unset All Classes The Melanie II User Manual TUTORIAL 2 ANALYZING GELS Now select the Region tool the third tool in the tool bar and draw a small rect angle in one of the gels using the mouse button To select groups in this region choose Select Groups In Region Choose Analyze Without Classes Set Differential Analysis Select the value type for example the feature area Accept the default value of the ratio 200 to be considered between the highest and the lowest values in groups See Differential analysis without classes set on page 13 4 To get a report on differential analysis results choose Show Analysis gt Report On Analysis You may print or save the report Correspondenc
23. s 94 0002 and 94 0005 and each time click Gels analyzed Y Macintosh HD Lippi 94 0005 mel ECOLI mel pairs Synthetic mel pen gels PLQEPE CEE 94 0002 mel Figure 16 4 The Open window allows the selection of the gels that will be opened and loaded into MelView Macintosh release on the Add button This selects the two gels and adds them to the Open gel box Click on the Done button The gels will be opened The Melanie II User Manual TUTORIAL 1 LOOKING AT GELS e In the Select menu choose the Gels Visible Gels option This will select all gels and draw a green line around each of them showing that they have been selected Fig 16 5 In MelView the green color always indicates selected objects So Melaniell Melview OR HA File Edit Select Show Stack Image Frocess Analjze DataBase Help la x Figure 16 5 The two opened gels Adjusting colors e Select the Hand tool the second tool from the top on the left hand side Put the cursor in one of the gels Hold down the right mouse button and move the gels until you see a region with many spots 1 On the Macintosh hold down the Command key 3 and the mouse button The Melanie II User Manual 16 5 16 TUTORIAL 1 LOOKING AT GELS e Now select the Region tool the third tool from the top in the tool bar and draw a small rectangle in one of the gels using the mouse button In the Select menu choose Select Mode Adjust Colors
24. t cd Jusr7 loceal Meleanierlil7Geals e Type the following command to run the MelView program gt MelView amp The amp sign indicates that the MelView program has to run in the background Figure 17 1 shows the screen that appears when running MelView i if qi Set meee Bees ie Dr i BIO RAD BEKELE Melaniell ee es The ie Lae ep ee eee or 2 pee ld dii Ain BELE EH Figure 17 1 The MelView main screen e Click on the Melanie II logo to get rid of it Setting preferences e This tutorial is also available on line To consult it on line Unix Melanie Il binaries PC Macintosh On the PC and Macintosh make sure that the Help directory is located in the same directory as the MelView application On 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 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 17 2 The Melanie II User Manual TUTORIAL 2 ANALYZING GELS Opening 2 DE images In Melanie II for PC PC e Click in the File menu and select the Open option The Open window will be displayed Fig 17 2 Hold down the Control key and click on each of the three 5 olecied fi LL rmai Figure 17 2 The Open window allows the selecti
25. the Select menu choose the Gels Visible Gels option This will select all gels and draw a green line around each of them showing that they have been selected Fig 17 5 In MelView the green color always indicates selected objects E Melaniell Melview Lof HAJ Eile Edit Select Show Stack Image Process Analyze DataBase Help la Figure 17 5 The three opened gels The Melanie II User Manual 17 5 17 TUTORIAL 2 ANALYZING GELS Adjusting colors e The 94 0002 and 94 0005 gels are raw not yet analyzed images ECOLI is an already analyzed master 2 DE map of Escherichia Coli from the SWISS 2DPAGE database In the Show menu choose Hide All to hide the already detected and annotated features in the ECOLI image e Select the Hand tool the second tool from the top in the left hand side tool bar Put the cursor in one of the gels Hold down the right mouse button and move the gels until you see a region with many spots e Now select the Region tool the third tool from the top in the tool bar and draw a small rectangle in one of the gels using the mouse and the button In the Select menu choose Select Mode Adjust Colors Select a logarithmic color function and adjust its parameter to the value 4 The change will directly be reflected in the selected region Fig 17 6 biri pami vic IO aj in ji ee ed E al lj Pr T hirea gaara E Loge 4 DE k Sm m Figure 17 6 The Adjust Colors option The modif
26. to the Internet you may directly access the SWISS 2DPAGE or SWISS PROT databases on the Expasy server as well as other databases on various servers 1 On the Macintosh use 4 instead 17 18 The Melanie II User Manual PC Unix I Macintosh i TUTORIAL 2 ANALYZING GELS Choose Database Set Master and choose ECOLI In the Unix release choose Database Set Browser and verify that the right name and path are given for Netscape Choose Database Set Server Enter www expasy ch and finally choose Database Set Database and enter SWISS 2DPAGE Using the Feature tool fifth tool on the tool bar select one feature correspond ing to an identified protein Then choose Database Query Server MelView will remotely control Netscape Navigator and request the corresponding entry from the SWISS 2DPAGE database on Expasy Analyzing data When gels have been matched with one reference gel groups have been cre ated and statistical data analysis may be performed on sets of features Close all gels that you have been using so far by choosing File Close and restart with another set of already analyzed gels of Plasma Perform the follow ing steps In Melanie II for PC or Unix Click in the File menu and select the Open option The Open window will be displayed Double click in the Gels Analyzed directory to open it Hold down the Control key and click on each of the six gel files called 94 0613 94 0614 94 0616 94 062
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