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ImageMaster 2D Platinum 6.0

1. 10 Select AT1bis and AT2bis using the Ctrl key and right click on one of their names Choose Open in the contextual menu 11 Apane is displayed for each class in the new Classes worksheet It is possible to display the master gel by choosing Show gt Show Master in the menu or using the F1 key The Master corresponds to the first common node in the match set structure that is the master gel of ImageMaster 2D Platinum Tutorial Edition AA 47 Data analysis match set Abis in the present case Select Show gt Show Master again or press the F1 key to make the Master disappear D ImageMaster 2D Platinum File View Edit Show Select Analyze Reports Tools Window Help aliilte e Workspace Project Bacteria phy ATI AT2 XB aA BTI i ai BT2 a5 Bacteria2 z d Abis d i Bbis Ca Classes Bacterialb ee T1 H a T2 A Bacterial A e BG AT e BG AT2 B Cd BT1 s B BT2 M Bacteria2 Bbis BT2bis M BT Ibis M Abis ne d A_T2_Gel3 mel N 4_T2_Gel2 mel Ja 3 Get mell Le Reports L Documents Please note that to compare gel populations the gels should have a common node in the match set that is used to construct the classes Without a common node the gels cannot be matched together and statistical comparison is impossible 4 4 Inter Class Report You are now ready to display an Inter Cl
2. Tutorial5 1 projects EI DIGE Gels Ki DIGE Gels ma Gel 01 ma Gel 02 ma Gel 03 d Gel 04 Cal MatchSets Lei Reports Ca Documents Gels 4 Spots 0 Annotations 0 5 8 Matching DIGE gels First position a landmark 1 Make sure all spots are deselected by first selecting all gel images Ctrl A and then choosing Select gt Unselect All in the menu if the option is grayed out all your spots were already deselected ImageMaster 2D Platinum Tutorial Edition AA 75 ED DIGE analysis Using the SHIFT key select the same spot on all gel images with the Spot tool choose small well defined unambiguously corresponding spots that are not in distorted regions Choose Edit gt Annotations gt Add Label in the menu Choose the category Landmark Click OK Supply a name such as L1 Click OK to close the window and Yes to confirm that you want to add labels to your gel images To perform automatic matching 6 7 10 Select all the images Ctrl A Choose Edit gt Matches gt Match Gels in the menu All the gels are matched to Master_DIGE When matching is completed ImageMaster gives the total number of matches found Click OK to close this message Choose File gt Save gt Worksheet to save all modifications annotations and pairs done in this worksheet To view the matching results 11 12 13 14 76 Show the match vectors between the
3. Match ID Max AT2bis Report Fron 173 0 133126 0 133126 0 133126 Ss eee eee E 13 In the newly created Inter Class Report choose Gap in the Displayed value list 14 Inthe main program window choose Window and select the previously saved report Inter class_Report_Ratio rpt from the list at the bottom of the menu It will pop up in front of the other report Close this report displaying the Ratios 15 Inthe remaining Gap report sort the values in descending order by clicking once in the Max column header 16 Click on the Annotate icon in the report toolbar to add an annotation category in the report Inter Class Report 2 CO Hei w o a ls e alei mames IE Inter Class Report Moll The chosen statistics are Mean 100 and Annotate 545 0 938567 0 938587 0 938587 0 915601 0 915601 0 915601 0 888545 0 888545 0 888545 0 812093 0 812093 0 812093 0 378539 0 378539 0 378539 0 316457 0 316457 0 316457 0 306124 0 306124 0 306124 54 ImageMaster 2D Platinum Tutorial Edition AA Data analysis 17 Enter the name of a new category called Set Verified Click OK and again OK to accept the default category constraints and properties 18 You will now have an extra column called Set Verified in your report 19 Select all rows in the gap report by clicking on the upper left cell in the table Inter Class Report 2 gei giel adale el m u Bl lee Inter Class Report v
4. 36 Select all the gels in the active worksheet Ctrl A 37 Choose Edit gt Annotations gt Copy Matched Labels in the main program menu 38 You will be asked if you want to propagate a certain number of labels from 1 category Set Verified to 6 gels Answer Yes 39 The software will tell you how many labels were added some spots are absent on certain gels so no labels are created in this case Click OK to close the window A0 All six gels will now have labels with the content Verified Save the gap report 41 Click on the Save icon in the toolbar of the gap report 42 Browse the folder C imageMaster 2D Platinum Tutorials Tutorial4 and save the report with the name Inter class_Report_Gap rpt in Report format Click Save The report is automatically included in the Reports folder of the project In order to confirm your results you can repeat this analysis with the classes AT1 and AT2 These contain the same gels but use matches from a different match set Bacteria1 In a next stage you may also want to compare classes Abis and Bbis To do so simply select these two classes and right click on one of them Choose Open from the contextual menu The twelve gels are opened in two panes as part of the classes Abis and Bbis and not as part of the four subclasses Additionally you can study the protein expression differences between the treatment groups T1 and T2 independently of the substrates substrate A and substrate
5. ImageMaster 2D Platinum Tutorial Edition AA 35 Spot detection and gel matching gel A_T1_Gel1 only appears if you choose Show gt Show Reference in the menu or press the F1 key D ImageMaster 2D Platinum Eek File View Edit Show Select Analyze Reports Tools Window Help Workspace Project Bacteria 2 Tutorial 1 projects F Bacteria Lei Gels Ca ari 4_T1_Gel3 mel 4_T1_Gel2 mel A TI Gell me AT2 A_T2_Gel3 mel A_T2_Gel2 mel 4_T2_Gell mel Cal MatchSets a d 4_T1_Gel3 mel 4_T1_Gel2 mel A TI Gell me Le Reports Lei Documents Gels 3 Spots 0 Annotations 0 8 Repeat the procedure steps 1 to 7 to create and open a match set AT2 which contains the three gels of AT2 in the Gels folder choose A_T2_Gel1 as a reference to create the Master 9 Close the open MatchSet worksheets by choosing File gt Close All in the menu In the following step you will create a higher level match set to allow the matching of populations AT1 and AT2 10 Right click on the MatchSets folder of the project and choose Create MatchSet in the contextual menu 11 Inthe Create MatchSet window enter A as a name for the new match set and click OK An empty match set A is created in the Match Sets folder 12 Drag and drop the match sets AT1 and AT2 onto A You are asked if you want to move them inside or after A Answer Inside 36 ImageMaster 2D Platinum Tutorial Edition AA Spot d
6. 0201256 tor cracanat 3 ia ss 1200o s000 207 070 es 0 153896 anzetend ze is 22000 4 2000 828 950 106002 0250000 eor cracow 4 sso 20 199000 3190000 209 190 seg 0 6179 FileName ae Intensity Area Vol Ratio Slope Gel 01 Cy3 Control 916 78 0000 3 92000 236 480 1 04300 0 357864 Uses e Lanser es ao 8 0000 320000 100310 5 80060 0 375022 72 The reported volume ratios Vol Ratio in the DIGE report are normalized so that the modal peak of volume ratios is zero since the majority of proteins are not up or down regulated This means that the Vol Ratio is expressed in the range of 1 to 1 000 000 for increases in spot volumes and 1 to 1 000 000 for decreases in spot volumes Values between 1 ImageMaster 2D Platinum Tutorial Edition AA DIGE analysis and 1 are not represented hence a two fold increase and decrease is represented by 2 and 2 respectively and not 2 and 0 5 as might have been guessed Please note that the DIGE report is the only occurence where the Vol Ratio is expressed this way 5 7 Creating DIGE match sets In the following section you will learn how to match the four DIGE gels of the project First a match set containing the four DIGE gels must be created 1 Inthe DIGE Gels folder of the Tutorial 5 project select the four DIGE gels not their individual images by clicking on their names while holding the Ctrl or Shift key 2 Right click o
7. 3 Inthe Add Gels In Class window enter AT1bis as a name for the created class and click OK ImageMaster 2D Platinum Tutorial Edition AA Data analysis Kl A Aclass called AT1ibis is created in the Classes folder Note that you can look at the MatchSet column in the File Details at the right side of the workspace window to see from which match set a gel is derived Bacteria2 Abis in this case It allows you to verify that the gels belong to the same match set which is necessary for statistical comparisons Workspace eH Project None E A_T1_Gel2 mel Bacteria2 abis ATibis EN A_r1_Geli mel BacteriaziAbis ATibis BT2 A Name MatchSet Class 3 i MatchSets ER 411 Gels mel Bacteriaziabis AT1bis ZA Bacteria 1 e A ad ATI H a AT2 kp A pm s BT2 d Bacteria2 d Abis amp _T1_Gel3 mel A_T1_Gel2 mel A TI Gell mel amp _T2_Gel3 mel A_T2_Gel2 mel A T3 Gell mel GA Classes Ma Bacterial aa A ma ATi ma AT2 B Ga Reports 7 La Documents 5 Repeat the procedure steps 1 to 4 to create the classes AT2bis BT1ibis and BT2bis always selecting the gels from the match set Bacteria2 ImageMaster 2D Platinum Tutorial Edition AA 45 Data analysis 46 Right click on the Classes folder and choose Create Class in the
8. ImageMaster 2D Platinum Tutorial Edition AA 25 Viewing and manipulating gels 3 Click on the Settings icon to choose the information to be displayed 4 Inthe pop up dialog box select attributes in the Visible list and click on the blue left pointing arrow to hide them and vice versa to show any Hidden attributes Click OK to confirm your choice These settings can be made permanent for a particular workspace by going to the Cursor Information tab in the ImageMaster Options choose Tools gt Options in the menu 5 Hold your cursor over the pixel of interest The Cursor Information window displays the desired information If the Value field shows Unloaded the raw data are not available In this case choose Edit gt Gels gt Raw Image gt Load 4 ImageMaster 2D Platinum A TT Dell no spot 15540 206 378 Gels 3 Spots 0 Annotations o 6 Close the Cursor Information window 26 ImageMaster 2D Platinum Tutorial Edition AA Spot detection and gel matching 3 Spot detection and gel matching 3 1 Introduction Spot detection in ImageMaster 2D Platinum was optimized to yield relevant biological results with minimum user interaction Only a few straightforward parameters need to be set to automatically locate the spots in a gel image Once this is done the gels can be matched possibly using one or two landmarks Note that this tutorial in particular describes the processing of non DIGE gels Most of the procedures a
9. Zoom the image to better see the left reference marker Pick the Annotation tool in the program toolbar Double click in the center of the reference marker Select the Comment category and click OK Enter IR1 as the label text and click OK The annotation is on the pixel where you double clicked If this is not the center of the reference marker you must move the annotation by clicking on its basis cross and dragging it to the middle of the marker You can now export a pick list with the included reference markers for each gel 23 24 25 Select spots to pick For example select the spots belonging to the set Pick by choosing Select gt Annotations gt By Category from the program menu and selecting Set Pick Click OK Choose File gt Export gt Spots to Picker gt GE Healthcare Ettan For each gel you will be asked to save a pick list in text or XML format only the text file can be read by the Ettan Spot Picker Enter a file name and destination folder and click Save The Ettan Spot Picker or Ettan Spot Handling Workstation can read the exported files You can include the pick list files in your workspace for later reference 26 27 28 29 62 Right click on the Documents folder under the Spot Picking project in the Tutorial4 Workspace Choose Add Files in the contextual menu Browse the appropriate folder select the pick lists and click OK The pick lists now appear in the Documents folder Ima
10. gt Shape gt Outlined in the menu det SA gt A aN A p e e og Se D O Crossed Outlined Filled Outlined Filled 28 Note that the default mode can be changed for a particular workspace by going to the Display tab in the ImageMaster Options choose Tools gt Options in the menu 3 5 Spot detection To detect spots efficiently we recommend previewing the spot detection results on a few small gel regions drawn with the Region tool before or during spot parameter optimization so that you can decide whether the parameters need to be fine tuned or not Each change in one of the spot detection parameters is immediately reflected in the selected region Once you find the optimal parameters you can detect spots on all selected gels To detect spots automatically 1 Select all gels Ctrl A 2 Drawa region with the Region tool on one or more selected gels in areas with representative spots 3 Choose Edit gt Spots gt Detect in the menu The Detect Spots window appears on the screen and the spots in the active regions are detected with the default parameters ImageMaster 2D Platinum Tutorial Edition AA Spot detection and gel matching ss 4 Adjust the Smooth parameter to detect all real spots and correctly split any overlapping ones a value of 2 works well with the tutorial gels At this level you should not be concerned with the high
11. 281747 0 263056 0 263056 0 263056 ojoj ujo an sj w n Gels 6 Spots 6 Annotations 0 0 250896 0 250896 0 250896 Mouse Selection 3D View v X 574 Y 355 Z 15582 X 553 Y 382 Z 10390 Iw Lighting I Spots overlay 32 H you now click on the Select Next icon in the toolbar of the Inter Class Report the spots of the next match are selected on the gels and directly shown in the 3D View window because you opened a mouse selection 3D View see details in the manual 58 ImageMaster 2D Platinum Tutorial Edition AA Data analysis Systematically verify all the matches in the gap report or stop when you consider having enough protein spots to start working with When finished label the interesting spots on the gels 33 Select all rows in the gap report by clicking on the upper left cell in the table 34 Click the Update Gels icon in the toolbar of the gap report 35 Answer Yes to add the labels in one gel the master gel EN imageMaster 2D Platinum File View Edit Show Select Analyze Reports Tools Window Help Gels 7 Spots 105 Annotations 17 Inter Class Report 2 On the master gel you will now see spots with the label Verified To easily mark the corresponding spots on the other gels as well so that they can be selected for spot picking for instance ImageMaster 2D Platinum Tutorial Edition AA 59 Data analysis 60
12. 417 3184 e Russia amp other C I S amp N I S Tel 7 095 232 0250 956 1137 Fax 7 095 230 6377 e South East Asia Tel 60 3 8024 2080 Fax 60 3 8024 2090 Spain Tel 93 594 49 50 Fax 93 594 49 55 e Sweden Tel 018 612 1900 Fax 018 612 1910 e Switzerland Tel 0848 8028 12 Fax 0848 8028 13 UK Tel 0800 616928 Fax 0800 616927 e USA Tel 800 526 3593 Fax 877 295 8102 imagination at work ProTang Teknikinformation AB 2005
13. Choose Reports gt 3D View in the menu ImageMaster displays a new 3D View window on top of the previous one It displays the 3D views of the active regions side by side Use the arrows at the right side of the window to simultaneously rotate all the gel regions Note that the different views are displayed with the same scale Therefore you can easily visualize any protein expression changes If your 3D views are centered on spots the heights of the different views may differ If you right click on the background in one of the views while holding the Shift key you will center all the views on this new position If this position also corresponds to background in the other gels the views will now be at the same height ImageMaster 2D Platinum Tutorial Edition AA Viewing and manipulating gels D ImageMaster 2D Platinum File view Edit Show Select Analyze Reports Tools Window Help 3D iew 2 IV Lighting I Spots overlay Gels 3 Spots 0 Annotations 0 3D View 3D View 2 16 You can visualize again the first 3D View window that displayed the single gel region To do so click on its tab at the bottom of the docked window 17 Close the 3D view windows 2 9 Cursor information To view information about pixels and spots once spots have been detected the Cursor Information window can be displayed 1 Choose Window gt Cursor Information in the menu 2 The Cursor Information window appears on the screen
14. Choose the default statistics Mean 100 and M S D 100 The sliders allow you to eliminate a certain percentage of outliers 100 means that all spots are included even outliers Leave the default value and click OK 7 Choose Ratio from the Displayed value list at the extreme right of the Inter Class Report toolbar 8 Click on the Save icon in the Inter Class Report toolbar to save your report 9 Browse the folder C imageMaster 2D Platinum Tutorials Tutorial5 and save the report with the name DIGE_Inter class _Report_Ratio rpt in Report format Click Save The report is automatically included in the Reports folder of the project 10 Choose the option Refine Selection from the Select on Gels drop down menu in the report toolbar 11 Choose the Max column and then click OK Note that you can choose one of the other columns Control or Treated However it is a good habit ImageMaster 2D Platinum Tutorial Edition AA DIGE analysis to select the Max column so that the operation is generic in particular for scripting purposes 12 Select rows having a Ratio higher than 2 then click OK 13 Make a new Report From Selection This feature can be accessed from the Reports drop down menu 14 Inthe newly created Inter Class Report choose Gap in the Displayed value list 15 Inthe main program window choose Window and select the previously saved report DIGE_Inter class_Report_Ratio rpt from the list at the bottom of the
15. MatchSets Classes Le Reports Ca Documents 1 5 Import gels into your project Now gel images must be incorporated into the Gels folder of your project 1 Right click on the Gels folder in the workspace navigator Choose the option Import Gels in the contextual menu 2 Inthe Import Image window select the input format of the files and indicate the reduction factor for example 2 to reduce the resolution from 300 to 150 dpi For the provided tutorial gels the reduction factor should be 1 and the file format TIFF Then click OK Import Image Please select the input format as well as the reduction factor Reduction factor TT RI File format ei coren 3 Browse the folder C imageMaster 2D Platinum Tutorials Tutorial1 and select the six TIFF images provided with this tutorial while holding the Ctrl key Click Open to import the gels A The imported images are saved with the extension mel You can change the file names manually automatically add an extension to all the a ImageMaster 2D Platinum Tutorial Edition AA 9 Experiment setup existing file names or decide to save the files in a different folder For the purpose of this tutorial just click OK to save the mel files in the proposed folder 5 Then enter the staining method that has been used to run this gel by choosing the appropriate one from the proposed list Gel Properties Specify gel properties Gel Name ago ell mel Staining Cancel The
16. Tutorial 1 and used in Tutorials 2 and 3 However as mentioned above additional gels and match sets have been added in the Tutorial4 workspace in order to demonstrate the power of the project organisation Choose Help gt Tutorials gt Tutorial 4 gt Restore in the menu to open this extended tutorial workspace 4 2 Open an existing workspace In case you work on your own gel files first open a workspace containing gels that were detected and matched please refer to Tutorials 1 and 3 to learn how to create a workspace with gels and match sets and how to carry out spot detection and gel matching 1 Display the Workspace window ImageMaster 2D Platinum Tutorial Edition AA 43 Data analysis 44 2 Click on the Open icon E in the toolbar of this window to open a workspace 3 Browse the appropriate folder and open your workspace file mws 4 3 Create classes In order to carry out comparisons of gels or gel populations classes have to be defined A class is a set of gels or gel populations with common biological properties that you can compare with other such entities Comparing classes will enable you to find the protein expression variations between different biological states To illustrate the use of classes in the ImageMaster project organisation two different match set structures have been created in the Tutorial 4 workspace In match set Bacteria1 the three gels from each subpopulation AT1 AT2 BT1 BT2 were first
17. exhibit statistically significant changes between control and treated groups of bacterial cultures Four replicate gels are loaded with bacterial lysates as indicated in the table below Gel number Cy2 Cy3 Cy5 Gel 1 Standard Control 1 Treated 1 Gel 2 Standard Treated 2 Control 2 Gel 3 Standard Control 3 Treated 3 Gel 4 Standard Treated 4 Control 4 Each gel contains a standard sample to normalize control and treated samples against The standard sample is derived from the control and treated lysates which are pooled in equal concentration Tutorial gels When you select Help gt Tutorials gt Tutorial 5 gt Restore in the menu any open worksheets and workspace are closed and the Workspace window containing an empty workspace is opened In this tutorial you can import the 12 GEL files 4 ImageMaster 2D Platinum Tutorial Edition AA 65 DIGE analysis DIGE gels 3 images each that can be found in C Program Files GE Healthcare lmageMaster 2D Platinum Tutorials Tutorial5 see directives below 5 2 Create a new workspace and project To create a new workspace 1 Click the Workspace tab below the ImageMaster toolbar to display the Workspace window Click on the New icon in the toolbar of the Workspace window If you work on the tutorial gels enter Tutorial5 in the Workspace Name field and browse the folder C imageMaster 2D Platinum Tutorials Tutorial1 to save your file You can e
18. gels Most of the procedures and functionalities will also apply to DIGE gels Please refer to Tutorial 5 to learn about the specificities of a DIGE gel analysis Tutorial gels When you select Help gt Tutorials gt Tutorial 1 gt Restore in the menu any open worksheets and workspace are closed and the Workspace window containing an empty workspace is opened In this tutorial you can import the 6 TIFF files that can be found in C Program Files GE Healthcare lmageMaster 2D Platinum Tutorials Tutorial1 see directives below 1 2 Display the workspace window The workspace is displayed in a dockable window like tabular reports and graphical reports Dockable windows enable you to manage the many documents that may need to be open while doing your analysis But whereas reports can be moved in different positions and closed at any time the Workspace window is always displayed at the left of the Gel Display Zone and cannot be closed due to its crucial role It can be in Pinned Auto Hide or Floating mode 1 Click the Workspace tab below the ImageMaster toolbar to display the Workspace window By default the Workspace window is displayed in Auto Hide Un pinned mode It will automatically hide or collapse when not in use to form a tab alongside the edge to which it is docked ImageMaster 2D Platinum Tutorial Edition AA Experiment setup Experiment setup D ImageMaster 2D Platinum File view Edit Show Select Analyze Reports Too
19. imported gel files now appear in the Gels folder of the project When you expand the Gels folder your workspace should look like the figure below Workspace Dl d Project None el Tutorial 1 projects E Bacteria Ga AT Gell mel 4_T1_Gel2 mel 4_T1_Gel3 mel 4_T2_Gell mel 4_T2_Gel2 mel amp _T2_Gel3 mel MatchSets Classes Le Reports Ca Documents The added gels can be grouped in subfolders within the Gels folder of the project 6 Right click on the Gels folder and choose the Create Folder option Enter AT1 as a name for the new folder 10 ImageMaster 2D Platinum Tutorial Edition AA Experiment setup Kl 7 Select the gels A_T1_Gel1 A_T1_Gel2 and A_T1_Gel3 while holding the Ctrl key and drag and drop them on the new AT1 folder 8 The subfolder AT1 now contains the three gels Repeat the procedure to create a subfolder called AT2 which contains the three other gels Workspace Deh Project None 7 SS Tutoriall 1 projects HEE Bacteria EN Ga Gels Avi ATI Gell mel 4_T1_Gel2 mel 4_T1_Gel3 mel E AT2 4_T2_Gell mel 4_T2_Gel2 mel 4_T2_Gel3 mel MatchSets Classes Le Reports L Documents Save the changes made to the project by clicking on the Save icon Ki in the workspace toolbar This does not save any changes made to your gels gels and workspace are independent and therefore need to be saved separately Please note that you can create as many projects
20. in the gap report Inter Class Report and press the Select on Gels icon in the report toolbar to see the corresponding spots on your gels If a spot belonging to the match is present on the gel it is displayed in the center of the cell in which the gel is displayed If it is absent in a gel you can activate the Hand tool and double click on the corresponding position in one of the other gels All gel images will be centered on this position Verify if the corresponding spots are properly matched If this is the case and if the spot is differently expressed in the two classes tick the check box in the Set Verified column of the Inter Class Report window To indicate that the report has been modified the corresponding cell becomes dark green or gray if the line was not selected and an asterisk appears after the window name Press the Select Next icon in the report toolbar to select the following match in the report and on the gels If the match is of interest that is if the spot is differently expressed check the corresponding box in the Set Verified column Sometimes a more quantitative view of the spots is necessary to decide whether a protein is differently expressed or not The Inter Class Intra Class Histograms are helpful in that case 26 27 28 Make sure the match you are interested in is selected in the gap report Use the Select on Gels Reports option This feature is available from the Select on Gels drop down me
21. its name and choosing Open in the contextual menu Again the reference gel is not open since it is automatically matched with the master of A You can match Master_AT2 against Master_A using the same procedure as for the matching of any other gel 42 ImageMaster 2D Platinum Tutorial Edition AA Data analysis 4 Data analysis 4 1 Introduction ImageMaster 2D Platinum offers several tools for examining gel data and finding protein expression variations This tutorial describes one of these analysis methods It provides a quick way to find significant expression differences between populations of gels Note that this tutorial in particular describes the processing of non DIGE gels Most of the procedures and functionalities will also apply to DIGE gels Please refer to Tutorial 5 to learn about the specificities of a DIGE gel analysis Since the exercise concentrates on the analysis of protein expression changes between populations of gels a larger set of sample gels is used In the following experiment four populations exist Cells were grown under two different conditions using substrate A and substrate B and underwent one of two treatments treatment 1 and treatment 2 Class Condition Treatment ATI Substrate A Treatment 1 A_T2 Substrate A Treatment 2 B_T1 Substrate B Treatment 1 B_T2 Substrate B Treatment 2 Tutorial gels For this tutorial you can continue working with the workspace created in
22. landmark annotations generally only one or two To position a landmark 2 Make sure all spots are deselected by first selecting all gels Ctrl A and then choosing Select gt Unselect All in the menu Using the Shift key select the same spot on all gels with the Spot tool choose small well defined unambiguously corresponding spots that are not in distorted regions Choose Edit gt Annotations gt Add Label in the menu Choose the category Landmark Click OK Supply a name such as L1 Click OK to close the window and Yes to confirm that you want to add labels to your gels EX imageMaster 2D Platinum Gels 3 Spots 3 Annotations 0 38 ImageMaster 2D Platinum Tutorial Edition AA Spot detection and gel matching To perform automatic matching 8 Select all the gels Ctrl A 9 Choose Edit gt Matches gt Match Gels in the menu 10 All the gels are matched to Master_AT1 11 When matching is completed ImageMaster gives the total number of matches found Click OK to close this message 12 Choose File gt Save gt Worksheet to save all modifications annotations and matches done in this worksheet To view the matching results 13 Show the match vectors between the spots in the front gel and those in the master by choosing Show gt Matches gt Show Vectors in the menu 14 Vectors are shown for all gels but the master gel These vectors are automatically minimized in the displayed region ImageMa
23. left cell in the table 36 Click the Update Gels icon in the toolbar of the Gap report 37 Answer Yes to add the labels to one gel the master gel 38 Click on the Save icon in the Inter Class Report toolbar to save your report 39 Browse the folder C imageMaster 2D Platinum Tutorials Tutorial5 and save the report with the name DIGE_Inter class_Report_Gap rpt Click Save The report is automatically included in the Reports folder of the project 40 Save all the changes by choosing File gt Save gt Save All in the menu 41 Close all the open reports by choosing Window gt Close All in the menu 42 Close all the open worksheets by choosing File gt Close All in the menu 5 11 Match with a preparative gel When a fluorescently post stained preparative gel is used for picking including reference markers it can be imported as a DIGE gel even if it is a single image This allows to use the same DIGE spot detection algorithm as for the corresponding DIGE gels The detected preparative gel can then be matched to the DIGE master to allow selection of spots to be picked 1 Right click on the DIGE Gels folder in the workspace navigator Choose Import DIGE Gel in the contextual menu 2 Inthe Import Image window select the input format of the files and indicate the reduction factor In this case the reduction factor should be 1 and the file format GEL Then click OK 3 Browse the folder C limageMaster 2D Platinum Tutorials Tutorial5 and sele
24. local GE Healthcare representative for the most current information Amersham Biosciences AB a General Electric company going to market as GE Healthcare Amersham Biosciences AB Bj rkgatan 30 751 84 Uppsala Sweden Amersham Biosciences Europe GmbH Munzinger Strasse 9 D 79111 Freiburg Germany Amersham Biosciences UK Ltd Amersham Place Little Chalfont Buckinghamshire HP7 9NA UK Amersham Biosciences Corp 800 Centennial Avenue PO Box 1327 Piscataway NJ 08855 1327 USA Amersham Biosciences KK Sanken Bldg 3 25 1 Hyakunincho Shinjuku ku Tokyo 169 0073 Japan Asia Pacific Tel 852 2811 8693 Fax 852 2811 5251 e Australasia Tel 61 2 9899 0999 Fax 61 2 9899 7511 e Austria Tel 01 57606 1619 Fax 01 57606 1627 e Belgium Tel 0800 73 888 Fax 03 272 1637 e Canada Tel 800 463 5800 Fax 800 567 1008 e Central East amp South East Europe Tel 43 1 982 3826 Fax 43 1 985 8327 e Denmark Tel 45 16 2400 Fax 45 16 2424 e Finland amp Baltics Tel 358 0 9 512 39 40 Fax 358 0 9 512 39 439 e France Tel 01 69 35 67 00 Fax 01 69 41 96 77 e Germany Tel 0761 4903 490 Fax 0761 4903 405 e Italy Tel 02 27322 1 Fax 02 27302 212 e Japan Tel 81 3 5331 9336 Fax 81 3 5331 9370 e Latin America Tel 55 11 3933 7300 Fax 55 11 3933 7304 e Middle East amp Africa Tel 30 210 9600 687 Fax 30 210 9600 693 e Netherlands Tel 0165 580 410 Fax 0165 580 401 e Norway Tel 815 65 555 Fax 815 65 666 e Portugal Tel 21 417 7035 Fax 21
25. matched together before creating match set A from AT1 and AT2 and B from BT1 and BT2 In match set Bacteria2 all six gels from population A were matched in a first step as were all six gels from population B The resulting match sets Abis and Bbis were then matched to give Bacteria2 Although these match sets use the same gels and detection the data analysis results may differ more or less depending on which match set is used for creating the classes to be analyzed In fact any of these match set structures can be justified and provide good results You can even use both to confirm your results or maybe find a few additional protein markers To show how this works the classes Bacteria1 and Bacteria1b have already been created in the Classes folder of the Bacteria project These two classes are based on the match set Bacteria1 Class Bacterial reproduces the organisation found in the corresponding match set A configuration as in class Bacteria1B rather allows you to compare all gels that underwent treatment T1 with those that were in group T2 You will now learn how to create a new class using the same structures as in class Bacteria1 but based on the match set Bacteria2 1 Inthe Workspace window expand the match set Abis within Bacteria2 and select the gels A_T1_Gel1 A_T1_Gel2 and A_T1_Gel3 by clicking on their names while holding the Ctrl key 2 Right click on one of the selected gels and choose Add In Class in the contextual menu
26. number of noise spots These are filtered out with the two other parameters 5 Choose Window gt Cursor Information in the menu Move your cursor over some spots that you consider to be noise or artifacts Look at the Saliency values for these spots AT Ge 2147482647 6254 0 r Parameters E Smooth al E gt 2 187 Min Area 4 lg 6258 AL A 5872 Saliency 50 0000 146 I Auto Preview Gels 6 Spots 0 Annotations 0 6 Enter a Saliency value in the spot detection window that is just above that of the spots to be filtered out All spots with Saliencies lower than the given threshold will be removed use a Saliency cutoff of 150 with the tutorial gels 7 Insome gels very small but intense artifacts for example dust particles remain detected and cannot be eliminated with the Saliency parameter without removing real spots Such artifact spots can be removed by ImageMaster 2D Platinum Tutorial Edition AA 29 Spot detection and gel matching setting an appropriate Min Area value expressed in number of pixels With the tutorial gels it is not necessary to use this parameter bul ImageMaster 2D Platinum 2147482724 e 12490 i Parameters pe Smooth FI si gt 2 164 Min Area 4 1 5 12522 A A 8855 Saliency 150 000 1709 V Auto Preview Cancel evie Gels 6 Spots 0 Annotations 0 8 When the detection preview is satisfactory click on OK to detect all spots in the selected gels
27. rights reserved mageMaster 2D Platinum uses the TIFF library 1988 1999 Sam Leffler and 1991 1999 Silicon Graphics Inc All rights reserved mageMaster 2D Platinum uses software developed by the Apache Software Foundation http www apache org 1999 2003 The Apache Software Foundation All rights reserved Cy CyDye Ettan Typhoon DeCuder ImageMaster LabScan and i i mageScanner are trademarks of GE Healthcare Ltd GE tagline and GE www a mersha m biosciences com monogram are trademarks of General Electric Company SYPRO is a trademark of Molecular Probes Inc GE Healthcare Macrovision is a registered trademark and FLEXIm is a trademark of Amersham Biosciences AB Macrovision Corporation ExPASy is registered by the Swiss Institute of Bi k t 30 Bioinformatics Microsoft Windows PowerPoint and Microsoft Internet JO f g atan Explorer logo are trademarks of Microsoft Corporation Netscape Navigator is a trademark of Netscape Communications Corporation TIFF and Photoshop are 75184U ppsala trademarks of Adobe Systems Incorporated Sweden All goods and services are sold subject to the terms and conditions of sale of the company within GE Healthcare which supplies them GE Healthcare reserves the right subject to any regulatory and contractual approval if required to make changes in specifications and features shown herein or discontinue the product described at any time without notice or obligation Contact your
28. tab at the bottom of the pane e To switch between stacked gels you can use the Page Up or Page Down keys on the keyboard e Double click with the Hand tool on a gel region to re center all the gels on the same position e To select all gels in a stack click on the pane banner e You can use View gt Pane Layout gt Free in the menu in order to define the number and the organization of cells columns and rows to display in the selected pane For instance if you choose to display the pane with 1 row and 3 columns you will get the following configuration ImageMaster 2D Platinum Tutorial Edition AA 17 Viewing and manipulating gels a ImageMaster 2D Platinum DER File view Edit Show Select Analyze Reports Tools Window Help I al e e Workspace Project Bacteria Tutorial 1 projects Bel Bacteria 4_T2_Gel3 mel A_T2_Gel2 mel B_T2_Gell mel MatchSets Classes Le Reports Ca Documents Gels 3 Spots 0 Annotations 0 NOTE In the same way panes can be stacked within a worksheet by using the different options of View gt Worksheet Layout Stacked Tiled Free in the menu e Move the legend of A_T1_Gel3 on the gel A_T1_Gel1 to invert their position in the pane Then choose View gt Switch or Ctrl F to cancel the previous operation and therefore to allow a quick switch between the two images e You can return to the original image organization by selecting View gt Pane
29. that these images have been matched automatically with their corresponding DIGE reference image having a red component 6 Right click on the match set DIGE and choose Open from the contextual menu 7 You are asked to define a Reference among the four DIGE gels to create the master for the match set DIGE Choose one of the gels with the most and best resolved spots For this tutorial choose Gel 03 and click OK 8 In the Workspace window the red component on the Gel 03 match set indicates that it is used as a reference to create the Master of match set DIGE 9 Four master images representing the four sub match sets are opened in a new MatchSet worksheet These master images Master_Gel 01 Master_Gel 02 Master_Gel 03 and Master Gel_04 are created based on the reference image Standard of each match set Note that instead of the Master_Gel 03 which is the Master of the reference sub match set 74 ImageMaster 2D Platinum Tutorial Edition AA DIGE analysis SI i Gel 03 the master Master_DIGE is shown As the master and reference images are the same and matches are automatically created between the two the reference image is not displayed by default It is possible to display it by choosing Show gt Show Reference in the menu To prevent confusion during matching we recommend not to display the Reference D ImageMaster 2D Platinum File View Edit Show Select Analyze Reports Tools Window Help Workspace Project DIGE
30. using the parameter values that were set 9 The spot shapes are displayed on the gels 10 Choose File gt Save gt Worksheet to save the spot detection results 11 Select the Region tool and double click to make the preview region disappear If holding the Shift key while doing this the preview regions from all gels are removed 12 Close the Cursor Information window 30 ImageMaster 2D Platinum Tutorial Edition AA Spot detection and gel matching 3 6 Selecting spots Spots can be selected with the Spot tool as long as they are visible on the gel Once selected the spots are highlighted in green e To select a spot make sure the Spot tool e activated and then click on the spot To select more than one spot select the first one and then hold the Shift key down while clicking on additional spots e Toselect all spots in a region position the cursor at the top left position of the desired region hold down the mouse button and then drag the cursor to the bottom right position e To deselect all spots select the gels for which you would like to deactivate the spots and click in one gel not on a spot e You can also select spots by using the options in the Select gt Spots menu 3 7 Editing spots Quantitative protein data and in particular the spot volume are highly dependant on an optimal and reproducible definition of the spot borders and a correct splitting of partially overlapped spots To increase the precisi
31. 9 0 378539 0 378539 I Sorted values 27 The corresponding spots are shown on the gels and the histogram for the selected match is highlighted in the nter Class ntra Class Histograms window If the values in the histogram clearly show that the protein is differently expressed in the two classes you can check the corresponding box in the Set Verified column of the Inter Class Report You can also display a 3D view for the corresponding spots on your gels 28 Select a line in your gap report 29 Click on the Select on Gels icon ImageMaster 2D Platinum Tutorial Edition AA 57 Data analysis 30 Choose Window gt Mouse Selection gt 3D View from the main program menu 31 A multiple 3D view is displayed see details in Tutorial 2 Since the views for the different gels use the same orientation and scale you can easily see the protein expression differences between the gels EX imageMaster 2D Platinum DAR File View Edit Show Select Analyze Reports Tools Window Help Inter Class Report File C Program Files GE Healthcar 2 Di A lg Blo a lele el o u B Inter Class Report voll The chosen statistics are Mean 100 and M S D BIBS YAO HA Match ID Max a AT2bis AT Ibis 409 1 08032 1 08032 1 08032 545 0 938567 0 938587 0 938587 0 888545 0 888545 0 888545 0 812093 0 812093 0 812093 0 378539 0 378539 0 378539 0 316457 0 316457 0 316457 0 306124 0 306124 0 306124 0 281747 0 281747 0
32. B used to grow the cells In this case you can work with the classes T1 and T2 Bacteria1b More generally you can design any class that helps you to carry out your analysis 4 5 Pick list To use the Ettan Spot Picker or Ettan Spot Handling Workstation two adhesive markers should be placed on the gel before scanning These markers are used for the calibration of the coordinates that is for determining the correspondence between the X and Y positions of the analyzed gel image and the coordinates of the actual gel located on the spot picker Once the gel has been digitized and analyzed with ImageMaster 2D Platinum the software can generate a pick list This list contains the location in pixels of the ImageMaster 2D Platinum Tutorial Edition AA Data analysis SSS center of each spot you wish to pick as well as the pixel coordinates of the centers of the two reference markers To export a file with spot coordinates for use by the spot picker you first have to open the image files and perform image analysis spot detection is mandatory You should then annotate the reference markers To do this exercise two detected gels with reference markers can be found in the Spot Picking project in the Tutorial4 workspace 1 Choose Select gt Gels gt All Ctrl A in the menu 2 Select File gt Close All in the menu 3 Select the gels SpotPick1 mel and SpotPick2 mel in the Gels folder of the Spot Picking project by clicking on th
33. GE Healthcare ImageMaster 2D Platinum 6 0 Tutorial Contents Tutorial Help 0 1 0 2 0 3 gg e CCU OMA E 1 Kgl IN 1 Starting Brel 2 1 Experiment setup 1 1 1 2 1 3 14 1 5 VERO elle de 3 Display the Workspace Window waeseecccssssssssssssssssssneesssssscscssssssssssnseessssseeeee 3 Cr ate d MEW AWOLKS PACES scania aceata tota aaa eds ci 6 PAINE WOOO CE aria sa ana ode doe 7 Import gels into your project o eessssssesesesssssscsssssssssssssssesssecsccsssssssssssseesesseees 9 2 Viewing and manipulating gels 2 1 2 2 2 3 24 2 5 2 6 2 7 2 8 2 9 ldrel elle de 13 Open an existing workspace wacesccccssssssssssssesesssecccsssssssssssesessesseccessssssssenees 13 Display gels in a worksheet 413 Hande gelni 18 Organize gels in panes Se Adjust CONtr AST ee Profile Ate IR Cursor information 3 Spot detection and gel matching 3 1 3 2 3 3 3 4 3 5 3 6 3 7 3 8 3 9 3 10 4 Data analysis 4 1 4 2 4 3 44 45 laude elt EE 27 Open an existing workspace oeiceccecsssssssssssssesssssscccssssssssssneesssssseesesssssssseeees 27 Open gels to Work E 27 Displaying spots EA Spot ELECTION EE Selecting SPOTS amenar inene ia 00 aa Editing EE SPOU FO PO EE Creating match sets Ga M tching Ve EE uge elle de 43 Open an existing workspace 43 Create classes 4 Inter Class Report 48 rekt 60 ImageMaster 2D Platinum Tutorial Edition AA Contents DIGE analysis 5 1 DEE DE Z
34. Layout gt Tiled in the menu 2 6 Adjust contrast Play with the contrast settings to get a feeling for your image quality 1 Select one or more gels 18 ImageMaster 2D Platinum Tutorial Edition AA Viewing and manipulating gels 2 Draw a preview region in one or more use the Shift key gels with the Region tool 3 Choose Show gt Gels gt Adjust Contrast in the menu It opens the Image Display Settings window Ay ImageMaster 2D Platinum Workspace Si Tutorial2 1 projects EF Bacteria ERE a 4_T1_Gell m 4_T2_Gel3 me 4_T2_Gel2 me 4_T2_Gell me MatchSets Classes Choose the color palette and set the contrast mapping options Le Reports P Colors Gray T Invert omen C Image 4_T1_Gel3 mel Unit Value X I Only in Region Bending 4 gt 0 Gels 3 Spots 0 Annotations 0 Cancel A Du moving the left or right borders of the slider below the histogram for the gel indicated in the Image field or by changing the Bending parameter you can modify the contrast and brightness of the image and see the effect on the selected regions 5 Checking the Only In Region box allows you to display the gray level histogram for the preview region only 6 Use the desired color option at the top of the window to modify the color palette Different predefined palettes are available Please note that ImageMaster 2D Platinum Tutorial Edition AA 19 Viewing and manipulating gels whatever
35. a MatchSets Classes Le Reports CA Documents 3 Select the pane called AT1 by clicking on its banner All the gels within this pane are now selected pane banner and gel legends in green whereas those from AT2 become unselected pane banner and gel legends in grey 4 Close the pane AT1 by choosing File gt Close Images in the menu Only the pane AT2 remains in the worksheet AT1 AT2 5 Open the AT1 gels again but in a different worksheet by selecting the sub folder AT1 in the workspace right clicking on it and choosing Open gt In New Worksheet A new worksheet called automatically AT1 is created and it appears at the front of the Display Zone while the previous worksheet is now inactive banner in grey Please note that there is always just one active worksheet 14 ImageMaster 2D Platinum Tutorial Edition AA Viewing and manipulating gels D ImageMaster 2D Platinum DER File view Edit Show Select Analyze Reports Tools Window Help alele Workspace Project Bacteria Tutorial2 1 projects El Bacteria 4_T2_Gel3 mel 4_T2_Gel2 mel A T3 Gell mel MatchSets Classes Ga Reports La Documents Gels 3 Spots 0 Annotations 0 To change the active worksheet 6 Switch the worksheets by clicking on the banner of AT1 AT2 To close a worksheet 7 Select all the gels displayed in worksheet AT1 AT2 with Select gt Gels gt All in the menu 8 Close them with File gt Close Images in t
36. abcdefghijkl abcdefghijkl 76 466 473 76 466 473 Normal Ratio 3 08088 2 58183 3 24276 Overlap Ratio 1 93352 1 89343 2 67644 Overlap Gap 0 0983950 0 0832530 0 102301 Negative values mean that the spots are under expressed in the second class when compared with the first one Positive values indicate that the spots are over expressed in the second class compared to the first class ImageMaster 2D Platinum Tutorial Edition AA 49 Data analysis 50 The groups 461 496 and 111 have a Normal Ratio ratio between the mean values for each class close to 6 However their class intervals may overlap entirely match 111 or partially match 461 On the other hand matches with lower Normal Ratios such as matches 76 466 and 473 can be much more interesting The individual values are quite homogenous within each class and there is no overlap between the two classes The overlapping measures Ratio and Gap as calculated in ImageMaster 2D Platinum are more powerful to find protein expression changes You can find details and schemes in the user manual In short and by slightly simplifying Ratio Overlap Ratio Ratio between the lower limit of one class interval class with the highest mean value and the upper limit of the other class interval class with the lowest mean value Absolute values smaller than 1 indicate overlap whereas absolute values higher than 1 show that there is no overlap between the class ranges If o
37. and view the signal intensity adjust contrast profile 3D view e Tutorial 3 Spot detection and gel matching Become skilled at performing automatic spot detection and matching on your gel images find the corresponding proteins in different gels e Tutorial 4 Data analysis Discover a powerful method to quickly find significant protein expression variations between two populations of gels and learn how to export a spot picking list e Tutorial 5 DIGE analysis Learn how to carry out a complete analysis of DIGE gels using an internal standard importing DIGE images observing variations within DIGE gels matching different DIGE gels and finally analyzing the protein expression variations ImageMaster 2D Platinum Tutorial Edition AA 0 3 Starting a tutorial When you choose a particular tutorial from the Help menu the software will close the previously opened images and open the appropriate tutorial workspace and files for the exercise Each time you do this the software restores the required files from an archive that cannot be overwritten and saves them in a particular folder In this way you can never destroy the original tutorial files For each tutorial you can restore the following states Help gt Tutorials gt Tutorial X gt Option e Restore Closes any open files and then restores the recommended files to start the tutorial with The tutorial instructions are displayed in a browser window that opens up a
38. as you want in your workspace You may also add existing projects right click on the workspace name choose Insert Project in the contextual menu browse the folder where the project file is located and select it file with a prj extension Later when gel matching must be performed match sets are created within the workspace see Tutorial 3 then classes are defined to carry out statistical analysis see Tutorial 4 If you select gels in the workspace you will see that detailed information about selected files is given on the right side of the Workspace window The details include the Name MatchSet and Class to which the image belongs dates Created and last Modified Size in bytes File Type File Path and ID ImageMaster 2D Platinum Tutorial Edition AA 11 Experiment setup 12 ImageMaster 2D Platinum Tutorial Edition AA Viewing and manipulating gels d Viewing and manipulating gels 21 Introduction ImageMaster 2D Platinum offers many functionalities and tools to manipulate and view gels and their intensity profiles This tutorial familiarizes you with some of these features Tutorial gels For this tutorial you can continue working with the workspace created in Tutorial 1 Alternatively you can select Help gt Tutorials gt Tutorial 2 gt Restore in the menu This closes any open files and displays a workspace called Tutorial2 in the Workspace window 2 2 Open an existing workspace In case you work on your own
39. ass Report enabling you to find the protein expression variations between the classes AT1bis and AT2bis In the Inter Class Report you can display for every selected match the central tendency e g mean value and dispersion of the spot values in each class Moreover ImageMaster 2D Platinum computes overlapping measures between the class 48 ImageMaster 2D Platinum Tutorial Edition AA Data analysis ss intervals which are defined by the range Central value Dispersion Central value Dispersion The overlapping measures were implemented because the ratio between the mean values in each class often used by researchers to find protein expression changes is only a very partial measure to express the difference between two populations Matches characterized by a relatively high ratio between two classes can still have a lot of spot values that overlap This is illustrated by the histograms below which display the individual spot values orange bars in each class separated by a gray vertical line The horizontal blue line indicates the mean value for each class and the red lines define the dispersion intervals 0 4 03 0 2 0 1 D abcdefghijkl abedefghijkl abcdefghijkl 111 461 496 111 461 496 Normal Ratio 5 78514 6 71484 6 04695 Overlap Ratio 0 0 730560 1 12634 Overlap Gap 0 0721690 0 00982704 0 00766187 0 4 03 03 SS 02 02 0 2 ak 0 1 0 1 a a a abcdefghijkl
40. atinum Tutorials Tutorial5 and select the image files Gel 01 Cy2 Standard gel Gel 01 Cy3 Control gel and Gel 01 Cy5 Treated gel while holding the Ctrl or Shift key Click Open to import the gel images 4 The imported images are saved with the extension mel You can change the file names manually automatically add an extension to all the existing file names or decide to save the files in a different folder For the purpose of this tutorial just click OK to save the mel files in the proposed folder 5 Then enter the name of the DIGE gel Gel 01 and the dye chemistry minimal or saturation that has been used DIGE Min You can also add a comment concerning this DIGE gel Click OK SSS ImageMaster 2D Platinum Tutorial Edition AA 67 DIGE analysis Create DIGE Name Gel 01 Dye Chemistry DIGE Min gt Comment Cancel 6 In the next DIGE Gel Properties windows enter the dye used for each image by choosing the right one in the proposed list If the DIGE dye is mentioned in the file name the program gives it by default Click OK DIGE Gel Properties Specify DIGE gel properties Gel Name Gel 01 Cy2 Standard mel Staining Cy2 Cancel 7 Select the reference gel image among the different images of the DIGE gel Obviously when an internal standard is used often with Cy2 it should be defined as the reference Click OK DIGE Reference Select the reference gel Gel 01 Cy2 Standard mel Gel 01 C
41. contextual menu In the Create Class window enter Bacteria2 as a name for the new class Now right click on the class Bacteria2 and choose Create Class in the contextual menu In the Create Class window enter Abis as a name for the new class Repeat this procedure to create a class Bbis within the class Bacteria2 Select the classes AT1bis and AT2bis by clicking on their names while using the Ctrl key and drag and drop them in the empty class Abis Answer Inside when asked to move the classes Inside or After class Abis Select the classes BT1bis and BT2bis by clicking on their names while using the Ctrl key and drag and drop them in the empty class Bbis Answer Inside when asked to move the classes nside or After class Bbis ImageMaster 2D Platinum Tutorial Edition AA Data analysis Workspace Dy bel Project None B12 cec me leg EN e 72 cerne escerezete orz Boss escerezete ere Boss Bacterozieos orie Busse aran EN A2 Semel Bacterazabs az EN A2 cerne Bacteraziabe aran BAT Sebel Bacterazabs Los BAT Seel Bacteraziabe Los EN nr cer nai escerazee ait 4_T1_Gel2 mel A TI Gell mel 4_T2_Gel3 mel A_T2_Gel2 mel A T3 Gell mel 1_Gel3 mel _T1_Gel2 mel H T T1_Gell mel T2_Gel3 mel T T 2 Gel2 mel 2_Gell mel E Ga Classes M Bacterialb Bacteria2 Name MatchSet Class d Abis E B_72_cela mel Bacteria2 ebis BT2bis 4_T1_Gel3 mel
42. ct the image file Pick gel Click Open to import the gel image A Click OK to save the mel file in the proposed folder 5 Then enter the name of the DIGE gel Pick You can also add a comment concerning this pick gel Click OK 6 Inthe next DIGE Gel Properties windows choose Sypro Ruby in the proposed list Click OK Beet ImageMaster 2D Platinum Tutorial Edition AA 85 DIGE analysis 86 GN 10 11 12 Workspace ki Project DIGE H Gels AS DIGE Gels hy Gel 01 phy Gel 02 hp Gel 03 phe Gel 04 vis Pick DN Pick mel L atchSets DIGE d Gel 01 Gel 01 Cy2 Gel 01 Cy3 Gel 01 Cy5 d Gel 02 Gel 02 Cy2 Gel 02 Cy3 Gel 02 Cy5 KE Gein Right click on the Pick DIGE gel and choose Detect in the contextual menu The gel image is loaded and opened in a new worksheet In the DIGE Spot Detection window enter an estimation of the Number of Spots present on the image This detection parameter generally should be close to the one used for the other DIGE gels Choose 1500 and click OK Save the changes made on the pick gel by choosing File gt Save gt Worksheet in the menu In the workspace navigator drag and drop the image Pick mel within the match set DIGE Right click on Pick mel within the match set DIGE and choose Open in the contextual menu This opens Pick mel and the master of the match set DIGE in a MatchSet worksheet Select the two gels I
43. cted spots To display this report 1 Select the gels containing the spots you are interested in 2 Select spots with the Spot tool or menu options 3 Choose Reports gt Spot Report gt Current Template A Spot Report is displayed A Click on the Settings icon to choose the information to be displayed In the pop up dialog box select attributes in the Visible list and click on the blue left pointing arrow to hide them and vice versa to show any hidden attributes Click OK to confirm your choice Note that you can include attributes from the master gel see section below on matching SSS 32 ImageMaster 2D Platinum Tutorial Edition AA Spot detection and gel matching E ImageMaster 2D Platinum Spot Report Settings Set the spots attributes to be displayed in the report Gels aoeds MOMA Visible attributes FileName Id Intensity Intensity 4 amp _T1_Gel3 8ef46743 1ac3 40ba 82e8 912340162 772 500 1 95000 519 145 0 053769 A_T1_Gel3 8ef46743 1ac3 40ba 82e8 912340162 1192 50 1 00000 522 550 0 083003 A_T1_Gel3 8ef46743 1ac3 40ba 82e8 912340162 2959 50 1 83000 1754 69 0 20599 A_T1_Gel3 8ef46743 1ac3 40ba 82e8 912340162 799 500 2 04000 515 290 0 055649 A T1 Gel3 8ef46743 1ac3 40ba 82e8 912340162 418 500 See 145 660 Bes G Spot Report Several features are listed below that apply to the Spot Report and in general to all other ImageMaster reports e A Spot Report ca
44. d analyzed during the course of a specific gel study To add a new project to your workspace 1 Right click on the workspace name Tutorial1 at the top of the workspace navigator 2 Choose the option New Project from the contextual menu Sa ImageMaster 2D Platinum Tutorial Edition AA 7 Experiment setup Workspace ee EAT Insert Project Backup Restore Workspace Restore Project Properties 3 Enter a new project name a destination folder for the project file Lol and a comment that describes the project For the purpose of this tutorial use the name Bacteria the same folder as before and enter a comment as indicated below Click OK Create New Project Enter New Project properties Project Name Bacteria Location ImageMaster 2D Platinums Tutorials Tutorial Comment In this project bacteria were cultivated either with substrate A or with substrate B In both growing conditions two treatments have been tested You find that your workspace now includes a project called Bacteria containing five default folders Gels MatchSets Classes Reports and Documents Please note that if you purchased a license for ImageMaster 2D Platinum DIGE a subfolder called DIGE Gels exists in the Gels folder See chapter 5 of these tutorials to learn how to use this DIGE Gels folder ImageMaster 2D Platinum Tutorial Edition AA Experiment setup ss Workspace Ci bal Project None
45. ding to Control samples by clicking on their names while holding the Ctrl key 2 Right click on one of the selected gels and choose Add In Class in the contextual menu ImageMaster 2D Platinum Tutorial Edition AA DIGE analysis 77 DIGE analysis SSS 3 Inthe Add Gels In Class window enter Control as a name for the created class and click OK 4 Aclass called Control is created in the Classes folder 5 Repeat the procedure steps 1 to 4 for the Treated samples 6 Select the classes Control and Treated by clicking on their names while using the Ctrl key 7 Right click on one of them and choose Open in the contextual menu 8 A pane is displayed for each class in the new Class worksheet It is possible to display the Master by choosing Show gt Show Master in the menu or using the F1 key SSS 78 ImageMaster 2D Platinum Tutorial Edition AA DIGE analysis D ImageMaster 2D Platinum File view Edit Show Select Analyze Reports Tools Window Help Workspace Project DIGE phy Gel 02 phy Gel 03 phy Gel 04 Cal MatchSets py DIGE a 01 Cy2 Standard 01 Cy3 Control mel Gel 01 Cy Treated mq 02 Cy2 Standard Gel 02 Cy3 Treated m 02 Cy5 Control mel 03 Cy2 Standard 03 Cy3 Control mel Gel 03 Cy5 Treated ma 04 Cy2 Standard Gel 04 Cy3 Treated mg 04 Cy5 Cantrol mel Gel 03 Cy3 Control mel Gel 01 Cy3 Control mel f Gel 02 Cy5 Control mel f Gel 04 C
46. e aa aaa eae tac aa aaa tata say dace aaa 65 5 2 Create a new workspace and Groe 66 5 3 FER Ee Eer 67 54 COALS CLING DIGE gels ebessi 69 55 Displaying a DIGE histogram iii 70 5 6 Displaying a DIGE report 12 5 7 Gr AtinG DIGE MACH StS EES 73 58 MotchneDlGkoaelg tege eg 75 5 9 Creating classes with DIGE IMG eS sssesseessssssccssssssssssssseseessessesssssssnsees 77 5 10 Comparing classes With DIGE images weecccscccssssssssssssesessssecccsssssssnees 79 5 11 Match with a preparative gel 5 12 Pick list ImageMaster 2D Platinum Tutorial Edition AA Tutorial Help 0 1 Introduction The ImageMaster 2D Platinum tutorials offer a step by step guide to analyzing your gel images and reporting results Once you are familiar with the basic workflow you can refer to the user manual for instructions on how to use additional tools and adapt procedures to your specific needs These tutorials are provided with gel images and related files enabling you to perform an entire gel analysis while highlighting the main features of the program Of course you can immediately carry out the analysis on your own gel files 0 2 Structure The following steps are illustrated with dedicated tutorials e Tutorial 1 Experiment setup Find out how to create a workspace to organize your experiment and how to import gels in projects e Tutorial 2 Viewing and manipulating gels Learn how to manipulate gel images select move zoom stack
47. e contrast of the open images using Show gt Gels gt Adjust Contrast in the menu see Tutorial 2 EE ImageMaster 2D Platinum Tutorial Edition AA 69 DIGE analysis D ImageMaster 2D Platinum File View Edit Show Select Analyze Reports Tools Window Help 70 DoR Gels 12 Spots 0 Annotations 0 5 5 Displaying a DIGE histogram When two co detected images are selected a DIGE histogram can be displayed It shows data associated with detected spots in the selected images Spot data is plotted against log volume ratio on the X axis using two Y axes e The left Y axis displays the spot frequency The blue curve represents the frequency distribution of the log volume ratios e The right Y axis represents the Measure parameter Area Max Volume Max Intensity or Max Slope selected in the dropdown menu in the toolbar of the DIGE histogram window A plotted single data point on the histogram represents an individual protein spot ImageMaster 2D Platinum Tutorial Edition AA DIGE analysis To display a DIGE histogram 1 Select two co detected images Gel01 Cy3 Control and Gel01 Cy5 Treated by clicking on their legend while pressing the Ctrl key 2 Select the spots to be included in the DIGE Histogram Generally you would select all spots in the images So choose Select gt Spots gt Allin the menu or use the Shift A shortcut 3 Choose Analyze gt DIGE gt Histogram in the menu A ADIGE h
48. e of outliers 100 means ImageMaster 2D Platinum Tutorial Edition AA Data analysis that all spots are included even outliers Leave the default value and click OK In the displayed window you will see four columns The first one displays the IDs of the matches i e the ID of the spot in the master gel The columns AT1bis and AT2bis contain the mean values in terms of Vol of all the spots in the corresponding class for the particular match The Max column displays the highest value of the two means You can change the content of the columns when you select a different measure in the Displayed value list at the top right corner of the Inter Class Report window When you choose Dispersion for example the values displayed in the columns AT1bis and AT2bis correspond to the dispersion M S D in each match Again the Max column displays the higher of the two values ImageMaster 2D Platinum Tutorial Edition AA 51 Data analysis D ImageMaster 2D Platinum Eile View Edit Show Select Analyze Reports Tools Window Help SEN A Gels 6 Spots 2506 Annotations 6 Inter Class Report Mal sel aide pl m u a Inter Class Report voll The chosen statistics are Mean 100 and M S D Max AT2bis ATibis Normalized 0 0344612 0 00562937 leet Auer 3 oooezae lee omoes 6 Choose Ratio from the Displayed value list at the right of the Inter Class Report toolbar 7 Click on the Save icon i
49. e report and on the gels 24 If the match is of interest that is if the spot is differently expressed check the corresponding box in the Set Verified column Sometimes a more quantitative view of the spots is necessary to decide whether a protein is differently expressed or not The Inter Class Intra Class Histograms are helpful in that case 25 Make sure the match you are interested in is selected in the gap report 26 Use the Select on Gels Reports option This feature is available from the Select on Gels drop down menu in the Inter Class Report toolbar Please note that the histogram window needs to be docked to remain visible see Tutorial 1 to learn more about docking report windows 56 ImageMaster 2D Platinum Tutorial Edition AA Data analysis D ImageMaster 2D Platinum File view Edit Show Select Analyze Reports Tools Window Help n x Inter Class Intra Class Histograms 4 X lg a le m ul Inter Class Histograms voll The chosen statistics are Mean 100 and aoeds 440 abcdef Geb 6 Spots 6 Annotations 0 Inter Class Report 2 Hei giele ele am n u B le Select on Gels nter Class Report Repat Select on Gels Reports The chosen statistics Match ID Refine Selection i Set Verified v 409 Select from Gels 1 08032 0 915601 0 915601 0 915601 i 481 0 888545 0 868545 0 888545 0 812093 0 812093 0 812093 I Adaptive gradations 4 a o o 0 37853
50. e the left reference marker 11 Pick the Annotation tool in the program toolbar 12 Double click in the center of the reference marker 13 Select the Comment category and click OK Beet ImageMaster 2D Platinum Tutorial Edition AA 87 DIGE analysis 14 Enter IR1 as the label text and click OK 15 The annotation is on the pixel where you double clicked If this is not the center of the reference marker you must move the annotation by clicking on its basis cross and dragging it to the middle of the marker 16 Repeat steps 10 to 15 for the right marker but enter IR2 instead of IR1 as a label for the annotation You can now export a pick list including the reference markers 17 Make sure that only the pickgel is selected 18 Select the spots belonging to the set Pick by choosing Select gt Annotations gt By Category from the program menu and selecting Set Pick Click OK 19 Choose File gt Export gt Spots to Picker gt GE Healthcare Ettan 20 You will be asked to save a pick list in text or XML format only the text file can be read by the Ettan Spot Picker Enter a file name and destination folder and click Save The Ettan Spot Picker or Ettan Spot Handling Workstation can read the exported files You can include the pick list files in your workspace for later reference 21 Right click on the Documents folder in the Tutorial5 Workspace 22 Choose Add Files in the contextual menu 23 Browse to the appropriate folder se
51. eir names while holding the Ctrl key A Right click on one of the gel names and choose Open gt In Worksheet in the contextual menu 5 Hide the workspace 6 Identify the two reference markers If the reference markers are well detected nice round spots perfectly centered on the marker during the automatic spot detection process you can just add an annotation on each of the marker spots This is the case for both reference markers in SpotPick1 mel and for the right marker in SpotPick2 mel 7 Zoom the image SpotPick1 mel to better see the left reference marker 8 Dick the Annotation tool in the program toolbar 9 Double click on the marker spot 10 Select the Comment category and click OK 11 Enter IR1 as the label text and click OK 12 The annotation will be linked to the marker spot and its coordinates will be those of the center of the spot 13 Repeat the procedure for the second right reference marker but enter the label text IR2 14 Repeat the procedure for the right reference marker in SpotPick2 mel Its label must be IR2 Beet ImageMaster 2D Platinum Tutorial Edition AA 61 Data analysis If the reference markers are not well detected irregular shape or split as is the case for the left reference marker in SpotPick2 mel d 16 17 18 19 20 21 22 Select the spot that is on the marker using the Spot tool Choose Edit gt Spots gt Delete Answer Yes when you are asked for confirmation
52. etection and gel matching 13 The match sets AT1 and AT2 should now be in match set A 14 Right click on match set AT1 and choose Set MatchSet as Reference in the contextual menu The match set AT1 gets a specific icon with a red component and a tick mark to show that its master image will be used to create the master of match set A D ImageMaster 2D Platinum File View Edit Show Select Analyze Reports Tools Window Help allee Workspace Project Bacteria El Tutorial 1 projects Bacteria Lei Gels E ATI AT Gei 4_T1_Gel2 mel A TI Gell me G AT2 p A_T2_Gel3 mel A_T2_Gel2 mel A_T2_Gell mel Cal MatchSets Ga Classes Le Reports Lei Documents Gels R Spots o Annotations o 3 10 Matching gels You can now effectively match first the gels in each sub match set then the match sets AT1 and AT2 First open match set AT1 1 In the Workspace window right click on the match set AT1 and choose Open in the contextual menu Note that by default the reference A_T1_Gel1 is not visible It doesn t need to be displayed since it is automatically matched with the master You can display the reference gel by choosing Show gt Show Reference or F1 key in the menu ImageMaster 2D Platinum Tutorial Edition AA 37 Spot detection and gel matching ImageMaster was designed to match gels with minimum user intervention but sometimes you may have to help the matching process by specifying a few
53. g with the Hand tool in this gel moves all the other gels even deselected ones to the same location and with the same zoom factor e Toselect gels to work on you can activate several images by holding down the Ctrl key as you click on their legends You can also select several gels by clicking on the first gel legend and then while holding down the Shift key clicking on the legend of the last gel in a desired series All gels in between become selected e Toselect all the gels in the active worksheet choose Select gt Gels gt All in the menu or use the Ctrl A keyboard shortcut 2 5 Organize gels in panes You can stack gels within a pane putting them one on top of the other This keeps a reasonable display size for each gel when working with many gels To stack images and manipulate image stacks e Make sure the pane is selected by clicking on its banner if necessary Choose View gt Pane Layout gt Stacked in the menu All the gels of the pane are now stacked 16 ImageMaster 2D Platinum Tutorial Edition AA Viewing and manipulating gels E ImageMaster 2D Platinum DER File Yiew Edit Show Select Analyze Reports Tools Window Help P Workspace Project Bacteria Tutorial2 1 projects IF Bacteria 4_T2_Gel3 mel 4_T2_Gel2 mel 4_T2_Gell mel MatchSets Classes Le Reports Lei Documents Gels 3 Spots 0 Annotations 0 e A hidden gel in the stack can be made visible by clicking on its
54. geMaster 2D Platinum Tutorial Edition AA Data analysis Workspace f Tutorial 2 projects E Bacteria zx Ce Gels d MatchSets Classes Le Reports Ca Documents Spot Picking MatchSets Classes Ca Reports Documents SpotPick1 txt SpotPick2 txt ImageMaster 2D Platinum Tutorial Edition AA 63 Data analysis 64 ImageMaster 2D Platinum Tutorial Edition AA DIGE analysis ea 5 DIGE analysis 5 1 Introduction If you purchased ImageMaster 2D Platinum DIGE you will have access to all the functionalities needed to efficiently and accurately analyze your DIGE gel images ImageMaster 2D Platinum DIGE in particular features the co detection algorithm developed by the GE Healthcare DeCyder software team NOTE Please note that if the DIGE functions are absent or grayed out in the software your license is not valid for the DIGE module and you will not be able to carry out the following tutorial In this tutorial you will learn more about the specificities of a DIGE gel analysis Note that the general procedure and most of the functionalities are the same as those used for conventional 2 DE gels Therefore it is recommended to work through the previous tutorials to get familiar with the most commonly used features The present tutorial illustrates the analysis of a DIGE experimental design incorporating an internal standard with several replicate samples This tutorial describes how to find proteins that
55. he menu 2 4 Handle gels Within a worksheet you can manipulate a single gel image or several images at a time ImageMaster 2D Platinum Tutorial Edition AA 15 Viewing and manipulating gels e Tomove gels use the Hand tool ch from the toolbar and drag the gels to the desired location You can also use the sliders at the right and bottom edges of each image e With the Magnify tool Q you can either zoom in left mouse button or zoom out right mouse button You can also zoom by resizing the sliders at the right and bottom edges of each image If you only need to briefly examine some details you can use the Magnifying glass by shortly holding the Ctrl key while pressing one of the mouse buttons and moving your cursor over the interesting region e To select only a portion of the image activate the Region tool then click and define the interesting area The selection can be moved by clicking inside the region and dragging or resized by clicking on its borders or corners Deselect a region by clicking outside it Defining a region can be necessary for previewing contrast mapping or spot detection for cropping gels or just for selecting objects in the region Some uses are illustrated in the rest of the tutorials e When holding the Shift key while moving or zooming gels or defining gel regions the manipulations are carried out on all gels even deselected gels e After moving or zooming a particular gel double clickin
56. image files first open a workspace containing your gels created using the instructions in Tutorial 1 1 Click on the Workspace tab to display the Workspace window 2 Click on the Open icon Gin the toolbar of this window to open a workspace 3 Browse the appropriate folder and open your workspace file mws 2 3 Display gels in a worksheet Gel images are always opened in Worksheets Each worksheet has a banner containing its name and type enclosed in brackets The worksheet type corresponds to the name of the workspace folder from where the gels are extracted Gels MatchSets or Classes Please note that you cannot open gels from different folder types in a same worksheet Within a worksheet gel images are grouped in Panes according to their belonging to folders or sub folders in the project To open gels in a worksheet 1 Select the subfolders AT1 and AT2 in the Gels folder by clicking on their names while holding down the Ctrl key 2 Right click on one of the subfolder and choose Open gt In Worksheet in the contextual menu You can see that the six gels are displayed in two ImageMaster 2D Platinum Tutorial Edition AA 13 Viewing and manipulating gels panes one for each sub folder AT1 and AT2 The worksheet is named automatically AT1 AT2 by ImageMaster 2D Platinum DER File view Edit Show Select Analyze Reports Tools Window Help Workspace Project Bacteria Tutorial2 1 projects El Bacteri
57. iple 3D view is displayed see details in Tutorial 2 Since the views for the different gels use the same orientation and scale you can easily see the protein expression differences between the gels Dock the 3D View window EN imageMaster 2D Platinum File view Edit Show Select Analyze Reports Tools Window Help gt Mouse Selection view 3D Gel 01 Cy3 Control Gel02 CyS Control Wel 03 Cy3 Control Gel 04 Cy5 Control Gel01 Cy5 Treated Gel02 Cy3 Treated Gels 8 Spots 8 Annotations 0 Gel 03 Cy5 Treated Gel 04 Cy3 Treated Inter Class Report 2 HCH Ee D nter Class Report Vol Ratio The chosen statistics are Mean 100 and M S D Match ID Max a Control Treated 2 33294 2 33294 1 40200 1 31357 f Iw Lighting 1 26160 I Spots overlay Inter Class Intra Class Histograms Mouse Selection View 3D 34 If you now click on the Select Next icon in the toolbar of the Inter Class Report the spots of the next match are selected on the gels and directly shown in the 3D View window because you opened a mouse selection 3D View see details in the manual Systematically verify all the matches in the gap report or stop when you consider having enough protein spots to start working with When finished label the interesting spots on the gels 84 ImageMaster 2D Platinum Tutorial Edition AA DIGE analysis SSS 35 Select all rows in the gap report by clicking on the upper
58. istogram containing all the selected spots is displayed 5 Inthe drop down menu of the Settings icon select Max Volume D Measure Max Volume Gels Gel 01 Cy3 Control Gel 01 Cy5 Treated Gels 12 Spots 2488 Annotations 0 ImageMaster 2D Platinum Tutorial Edition AA 71 DIGE analysis DIGE Report alo alele e m B ul 5 6 Select several data points in the DIGE Histogram by using the Ctrl key or holding the left mouse button while drawing a blue region Click on the Select on Gels icon in the DIGE Histogram toolbar The corresponding spots will be selected on the images Displaying a DIGE report A DIGE report is a special case of a spot report enabling you to display volume ratios for specific combinations of DIGE images Two co detected images must be selected to generate a DIGE report To display a DIGE report for the spots selected in the previous section from the DIGE Histogram 1 Select the DIGE Report option from the Reports drop down menu in the toolbar of the DIGE Histogram window Alternatively if the two images Gel01 Cy3 Control and Gel01 Cy5 Treated and some spots are still selected you can choose Analyze gt DIGE gt Report in the menu A DIGE report containing only the selected spots is displayed You can use the Settings icon to display only the columns of interest see Tutorial 3 EE reso cracow 2 sor ateelselsacl 1 5199
59. lect the pick lists and click Open 24 The pick lists now appear in the Documents folder 88 ImageMaster 2D Platinum Tutorial Edition AA This version of ImageMaster has been developed by the Swiss Institute of Bioinformatics in collaboration with GeneBio and GE Healthcare All intellectual property rights on this Tutorial as well as on the ImageMaster 2D Platinum software belong to the Swiss Institute of Bioinformatics No part of this Tutorial may be reproduced or transmitted in any form or by any means electronic or mechanical including photocopy recording or any information storage or retrieval system without permission in writing from the Swiss Institute of Bioinformatics 1992 2005 Swiss Institute of Bioinformatics All rights reserved Swiss Institute of Bioinformatics CMU Rue Michel Servet 1 CH 1211 Geneva Switzerland ImageMaster 2D Platinum provides access to several databases on the Internet It is the responsibility of the user to acquire the database licenses if needed In particular the PROSITE and SWISS 2DPAGE databases are copyright and all commercial users of these databases are required to purchase a database license from Geneva Bioinformatics GeneBio SA Please contact GeneBio at info genebio com for more information Geneva Bioinformatics GeneBio SA Avenue de Champel 25 CH 1206 Geneva Switzerland mageMaster 2D Platinum uses the DeCyder co detection algorithm 2005 General Electric Company All
60. ls Window Help lol e Workspace z a 5 D O v Project None El ImageMaster D projects 2 You can click on the Collapse icon to hide the Workspace window 3 Click again on the Workspace tab to display the Workspace window To display the Workspace window in Pinned mode 4 Click on the Pin icon at the top right corner of the window The icon is changed to indicate that the window is now in Pinned mode In this mode the window is locked and therefore will not be hidden when not used You can move a Pinned window by dragging its title bar ImageMaster displays blue arrow guides to indicate where the window may be locked In the case of the workspace only the left position is allowed ImageMaster 2D Platinum Tutorial Edition AA Experiment setup D ImageMaster 2D Platinum File view Edit Show Select Analyze Reports Tools Window Help Hlafnfe Workspace Project None ZE ImageMaster D projects Gels o Spots D Annotations 0 Ay ImageMaster 2D Platinum File view Edit Show Select Analyze Reports Tools Window Help elalnfe Workspace Gels 0 Spots 0 Annotations 0 To display the Workspace window in Floating mode Drag the window title bar outside the blue arrow guides In this mode the Workspace window can be placed anywhere on your screen even outside the boundaries of the main program window Note that the Close icon is unavailable in the case of the Workspace window but i
61. mageMaster 2D Platinum Tutorial Edition AA DIGE analysis PE 13 Choose Edit gt Matches gt Match Gels in the menu 14 The image Pick is matched to Master_DIGE 15 When matching is completed ImageMaster gives the total number of matches found Click OK 16 Ifthe matching results are not satisfying see Tutorial 3 for details about matching validation you can add landmarks on the pickgel and the master and re run the matching process to improve it 5 12 Pick list 1 A pick list can now be generated 2 Choose Select gt Annotations gt By Category in the menu 3 Inthe Select Labels By Category window pick Set Verified in the category list Click OK 4 Choose Select gt Matches gt For Spots in the menu All spots in Pick mel matched to Verified spots in the Master_DIGE are selected Make sure the matches are correct 5 Unselect the master image by clicking on its legend while holding the Ctrl key 6 Choose Edit gt Annotations gt Add Label 7 Inthe Add Label window create a category Set Pick Click OK and again OK to accept the default category constraints and properties If the reference markers are not well detected as is the case for the present pick gel you can delete the spots and just define an annotation to mark the position of the reference markers 8 Select the spots that are on the markers 9 Choose Edit gt Spots gt Delete Answer Yes when you are asked for confirmation 10 Zoom the image to better se
62. menu It will pop up in one of the docking areas in front of the other report Close this report displaying the Ratios 16 Inthe remaining Gap report sort the values in descending order by clicking once in the Max column header 17 Click on the Annotate icon in the report toolbar to add an annotation category in the report 18 Enter the name of a new category called Set Verified Click OK and again OK to accept the default category constraints and properties 19 You will now have an extra column called Set Verified in your report Inter Class Report 2 Bal el ziel Six bk Inter Class Report Vol Ratio The chosen statistics are Mean 100 and M S D Match ID Max a Control Treated Set Verified ae orar soro aert ImageMaster 2D Platinum Tutorial Edition AA 81 ED DIGE analysis 82 20 21 22 23 24 25 Select all rows in the gap report by clicking on the upper left cell in the table Dock this report window to avoid that it closes automatically Pick the Inter Class ntra Class Histograms option from the Histograms drop down menu in the report toolbar This displays a window with histograms showing the individual spot values orange bars in each class separated by a gray vertical line The horizontal blue line indicates the mean value for each class and the red lines define the dispersion intervals Dock this histogram window to avoid that it closes automatically Select the first line
63. n be saved i printed 8 or selected lines in the report can be copied to the clipboard for pasting in other software such as MS Excel e Spots can be selected from reports by double clicking the corresponding line or by selecting the spot s in the report and clicking the Select on Gels icon CH in the report toolbar e One can also select and view the Next Y or Previous 4 spot on the gel e Thespotscanbe sorted according to one of the criteria displayed in the Spot Report by simply clicking on the header of the corresponding column ImageMaster 2D Platinum Tutorial Edition AA 33 Spot detection and gel matching by ImageMaster 2D Platinum DER File en Edt Show Select Analyze Reports Tools Window Help ad Yoh Spot Report Sal le aide el el ale Information on selected select on Gels FileName SpotID x Tv Intensity a Area Vol YeIntensity ol Saliency 4_T2_Gel2 1477 1034 20942 2 8 06000 82534 8 1 04859 1 51769 2727 16 A_T2_Gel 1380 1013 so 20763 0 43 4700 1 22887 9 32264 4275 67 3 4 5 Spot Report Report 5 Close the Spot Report window 6 Close the AT1 AT2 Gels worksheet by choosing File gt Close All in the menu 3 9 Creating match sets One of the major innovations of ImageMaster 2D Platinum 6 0 is the possibility to match populations of gels instead of only matching gels The population matching requires the definition of one or more match sets which include gel
64. n one of the selected gels and choose Create MatchSet in the contextual menu 3 Inthe Add DIGE in MatchSet window enter DIGE as a name for the created match set and click OK A Each of the following Create MatchSet windows corresponds to one of the four DIGE gels which will become sub match sets that contain their three respective images Keep the proposed names for all these match sets click four times OK ImageMaster 2D Platinum Tutorial Edition AA 73 5 DIGE analysis Workspace Dc ed Project DIGE f Tutorial 1 projects Name MatchSet vac BB Gel 01 Cy2 Standard mel DIGE Gel 01 els GA DIGE Gels FA Gel 01 Cy3 Control mel DIGE Gel 01 bi Gel 01 fil Gel 01 Cy5 Treated mel DIGE Gel 01 E ma e BR Gel 02 Cy2 Standard mel DIGE Gel 02 E d Gel 04 FA Gel 02 Cy3 Treated mel DIGE Gel 02 LR MatchSets RA Gel 02 Cy5 Control mel DIGE Gel 02 A Gel 03 Cy2 Standard mel DIGE Gel 03 Bi Gel 03 Cy3 Control mel DIGE Gel 03 FA Gel 03 Cy5 Treated mel DIGE Gel 03 D Gel 04 Cy2 Standard mel DIGE Gel 04 RA Gel 04 Cy3 Treated mel DIGE Gel 04 Lei Reports Ca Documents EA Gel 04 Cy5 Control mel DIGE Gel 04 5 You will find that a match set DIGE has been created in the MatchSets folder of the project and contains the four sub match sets Gel 01 Gel 02 Gel 03 and Gel 04 Expand the sub match sets by clicking on the H signs The red tick in the corner of the Control and Treated images indicates
65. n the Inter Class Report toolbar to save your report 8 Browse the folder C ImageMaster 2D Platinum Tutorials Tutorial4 and save the report with the name Inter class _Report_Ratio rpt in Report format Click Save The report is automatically included in the Reports folder of the project 52 ImageMaster 2D Platinum Tutorial Edition AA Data analysis 9 Choose the option Refine Selection in the Select on Gels drop down menu Inter Class Report File C Program Files GE Healthcare ImageMaster 2D Platinum Tutor D l Select on Gels Select on Gels Reports een zy of i Wee 10 Choose the Max column and then click OK Note that you can choose one of the other columns AT1bis or AT2bis However it is a good habit to select the Max column so that the operation is generic in particular for scripting purposes 11 Select rows having a Ratio higher than 2 then click OK Refine by Value Only keep selected the rows for which the Ma value verifies IV gt f2 _ lt Cancel 12 Make a new Report From Selection This feature can be accessed from the Reports drop down menu ImageMaster 2D Platinum Tutorial Edition AA 53 Data analysis Inter Class Report File C Program Files GE Healthcare ImageMaster 2D Platinum Tutor X lei elo alele 2l a u Bl ha a Gel Report Inter Class Report voll The chosen statistics are Mean 100 and M 5 D Intra Class Report
66. nd functionalities will also apply to DIGE gels Exceptions are the spot detection parameters and quantification values Please refer to Tutorial 5 to learn about the specificities of a DIGE gel analysis Tutorial gels For this tutorial you can continue working with the workspace created in Tutorial 1 and used in Tutorial 2 Alternatively you can select Help gt Tutorials gt Tutorial 3 gt Restore in the menu This closes any open files and displays the Workspace window with a workspace called Tutorial3 3 2 Open an existing workspace In case you work on your own image files first open a workspace containing gels created using the instructions in Tutorial 1 1 Display the Workspace window A Click on the Open icon GF in the toolbar of this window to open a workspace 3 Browse the appropriate folder and open your workspace file mws 3 3 Open gels to work on Open the gels to work on during this session 1 Select the subfolders A_T1 and A_T2 by clicking on their names while holding down the Ctrl key 2 Right click on one of the names and choose Open gt In Worksheet in the contextual menu ImageMaster 2D Platinum Tutorial Edition AA 27 Spot detection and gel matching 3 4 Displaying spots ImageMaster allows you to display spots in different ways Before starting spot detection it is best to change the spot shape to the Outlined mode 1 Select all the gels in the worksheet Ctrl A 2 Choose Show gt Spots
67. ne class interval is completely overlapped by another one you will find a O value for the Ratio Negative values indicate that the spots in the current class are under expressed compared to those in the other class NOTE In the rest of the tutorials Ratio will designate the Overlap Ratio as calculated by ImageMaster Please note that you can calculate the Normal Ratio by setting the dispersion percentage slider in the dialog to 0 see below Gap Overlap Gap Difference between the lower limit of one class interval class with the highest mean value and the upper limit of the other class interval class with the lowest mean value Negative values indicate overlapping intervals whereas positive values correspond to non overlapping class ranges You will find that sorting your matches based on these two criteria is a very efficient way to detect interesting protein expression changes Proceed as follows 1 Choose Select gt Gels gt All Ctrl A in the menu to select all gels Only selected gels will be included in the analysis 2 Select all matches by choosing Select gt Matches gt All in the menu 3 Choose Analyze gt Inter Class gt Report in the menu 4 Select the value type to be used In most non DIGE applications including this tutorial using the Vol is appropriate Click OK 5 Choose the default statistics Mean 100 and M S D 100 The sliders allow you to eliminate a certain percentag
68. nter a comment if you like Click OK when you are finished Right click on the workspace name Tutorial5 at the top of the workspace navigator and choose New Project in the contextual menu Enter DIGE as a new Project Name a destination folder Location for the project file prj and possibly a Comment that describes the project Click OK You will find that your workspace now includes a project called DIGE containing the folders Gels MatchSets Classes Reports and Documents If you purchased a license for ImageMaster 2D Platinum DIGE a subfolder called DIGE Gels exists in the Gels folder Workspace Za Dl Proiect None 66 2 TutorialS 1 projects Be DIGE C Gels CA DIGE Gels MatchSets Classes Ca Reports Ca Documents ImageMaster 2D Platinum Tutorial Edition AA DIGE analysis ss 5 3 Import DIGE gels The images of each DIGE gel must be imported in the DIGE Gels folder in order to create a DIGE gel entity 1 Right click on the DIGE Gels folder in the workspace navigator Choose Import DIGE Gel in the contextual menu 2 Inthe Import Image window select the input format of the files and indicate the reduction factor For the provided tutorial gels the reduction factor should be 1 and the file format GEL Then click OK Import Image Please select the input format as well as the reduction factor Reduction factor 4 ES File format GEL e 3 Browse the folder C mageMaster 2D Pl
69. nu If you observe mismatches you can delete bad matches or manually add new ones To edit matches e Delete bad matches by selecting the corresponding spot s and choosing Edit gt Matches gt Delete Match in the menu ImageMaster 2D Platinum Tutorial Edition AA 41 Spot detection and gel matching e Manually add matches by selecting the relevant spots and choosing Edit gt Matches gt Add Match in the menu e You can also add multiple pairs that is one spot in the master gel can be matched to several spots in another gel First select the spot in the master gel and then select hold the Shift key all the spots that should be matched with it Choose Edit gt Matches gt Add Match in the menu to add matches between all selected spots and the spot in the master gel thus forming a spot match The image below showing a corresponding region on the gels in tiled mode best illustrates the notion of multiple matches a single spot in the master gel corresponds to a double spot in A_T2_Gel2 EN imageMaster 2D Platinum DAR File view BS Show Select Analyze Reports Tools Window Help Undo Ctrl Z Redo Shift 2 History Gels Spots Annotations 5 Match Gels Match Annotations Add Match Ctel Shift G Delete Match Ctrl Shift U a gt 4 Gels 1 Spots 1 Annotations 0 19 Repeat the matching procedure steps 1 to 18 for match set AT2 20 Then open match set A from the workspace by right clicking on
70. nu in the Inter Class Report toolbar The corresponding spots are shown on the gels and the histogram for the selected match is highlighted in the Inter Class Intra Class Histograms window You may have to click on the docked tab of this window to bring it to the front You can also pin the window so that it remains visible see Tutorial1 for more details about dockable windows ImageMaster 2D Platinum Tutorial Edition AA DIGE analysis Kl 29 If the values in the histogram clearly show that the protein is differently expressed in the two classes you can check the corresponding box in the Set Verified column of the Inter Class Report E ImageMaster 2D Platinum DAR File view Edit Show Select Analyze Reports Tools Window Help Inter Class Histograms Vol Ratia The chosen statistics are Mean 100 and abcdefgh 1336 abcdefgh 1203 Spots 8 Annotations 0 Inter Class Report 2 Mei alle sei em Select on Gels nter Class Report Mi The chosen statistics Match ID Refine Selection fe abcdefgh 2 1273 Select from Gels j 2 70409 IV Adaptive gradations Sorted values You can also display a 3D view for the corresponding spots on your gels 30 Select a line in your gap report 31 Click on the Select on Gels icon 32 Choose Window gt Mouse Selection gt 3D View from the main program menu ImageMaster 2D Platinum Tutorial Edition AA 83 DIGE analysis 33 A mult
71. oll The chosen statistics are Mean 100 and M S D peeo E ANEI L 20 Pick the Inter Class Intra Class Histograms option from the Histograms drop down menu in the report toolbar This displays a window with histograms showing the individual spot values orange bars in each class Separated by a gray vertical line The horizontal blue line indicates the mean value for each class and the red lines define the dispersion intervals 21 Select the first line in the gap report Inter Class Report and press the Select on Gels icon in the report toolbar to see the corresponding spots on your gels If a spot belonging to the match is present on the gel it is displayed in the center of the cell in which the gel is displayed If it is absent in a gel you can activate the Hand tool and double click on the corresponding position in one of the other gels All gel images will be centered on this position 22 Verify ifthe corresponding spots are properly matched If this is the case and if the spot is differently expressed in the two classes tick the check box in the Set Verified column of the Inter Class Report window To indicate that the report has been modified the corresponding cell ImageMaster 2D Platinum Tutorial Edition AA 55 Data analysis becomes dark green or gray if the line was not selected and an asterisk appears after the window name 23 Press the Select Next icon in the report toolbar to select the following match in th
72. on is defined the software asks whether your 3D view should be based on the selected region or selected spots ImageMaster displays a 3D view for the active region or the area around the selected spots Pin the dockable 3D View window by clicking on the ial icon to show the 3D View beside the gels ImageMaster 2D Platinum Tutorial Edition AA Viewing and manipulating gels AM ImageMaster 2D Platinum DER File view Edit Show Select Analyze Reports Tools Window Help ala maint IV Lighting Gels 1 Spots 0 Annotations 0 Spots overlay 7 Byclicking on the arrows at the right side of the 3D view you can turn the 3D view in all directions or zoom in or out 8 Right click anywhere in the 3D view to center the view on the clicked position 9 Youcan hide the axes in the view by selecting the Display Axes option under the Show icon in the 3D View window Repeat the operation to display the axes again The 3D View feature in ImageMaster enables comparative viewing of corresponding regions or spots in different gels To display multiple 3D views of several matching gel regions ImageMaster 2D Platinum Tutorial Edition AA 23 Viewing and manipulating gels 24 10 11 12 13 14 15 Use Shift to select the same region in all open gels using the Region tool Select the gels for which you would like to display and compare the 3D views select the three AT1 tutorial gels with the Ctrl A shortcut
73. on of quantitative work it is therefore highly recommended to create spots using the automatic spot detection algorithm in ImageMaster It is not advisable to manually edit spots because this introduces significant quantification errors Please refer to the ImageMaster user manual for instructions if you still prefer to edit spots yourself split merge grow and reduce You can of course delete spots 1 Select the spots to be deleted 2 Choose Edit gt Spots gt Delete in the menu Tips for spot editing e Make sure all real spots are detected and correctly split in the spot detection process If artifacts are detected you can always filter them based on Saliency Min Area or if this tends to remove also real spots based on spot quantification measures such as Intensity Volume Intensity Vol Area e Instead of splitting or merging spots when two spots in one gel seem to correspond to a single larger spot in another gel you can compare the summed quantification values of the two spots with the quantification value of the single spot To make such comparisons easier ImageMaster allows you to create and select multiple matches between one spot in the master ImageMaster 2D Platinum Tutorial Edition AA 31 Spot detection and gel matching gel and several spots on the other gels in the match set but not the inverse see the user manual for details 3 8 Spot report A Spot Report summarizes relevant information about the sele
74. orial2 1 projects Bel Bacteria 4_T2_Gel3 me A_T2_Gel2 me 4_T2_Gell me MatchSets Classes Le Reports Lei Documents Gels 3 Spots 0 Annotations 0 3 To deactivate the Profile feature untick Show gt Gels gt Profile in the menu 4 You can unload the raw image data to regain memory To do so select Edit gt Gels gt Raw Image gt Unload in the menu This is only necessary when your memory resources are low Before going on with the following section reorganize your gels once again ImageMaster 2D Platinum Tutorial Edition AA 21 Viewing and manipulating gels 22 5 2 8 Choose View gt Pane Layout gt Free in the menu and choose 3 rows and 1 column 3D view Another way to examine the intensity variations in a gel is by looking at the three dimensional 3D view of a gel region In such a view the isoelectric point pl and molecular weight MW values are as usual represented by the X and Y axes whereas the pixel intensity is plotted along the third dimension Z axis The resulting image shows a peak for each protein spot with a peak height that is proportional to the spot intensity To display a 3D view of a single gel region 1 2 Select the gel for which you would like to display the 3D view Select a region in this gel Alternatively you can select one or more spots using the Spot tool Choose Reports gt 3D View in the menu When spots are selected and moreover a regi
75. palette is chosen it can only be applied to all or none of the gels even those that are not selected or in a selected worksheet EN ImageMaster 2D Platinum o x Tutorial2 1 projects e EF Bacteria 4_T2_Gel3 me A_T2_Gel2 me 4_T2_Gell me MatchSets Classes Lei Reports Lei Documents Image 4_T1_Gel3 mel z Unit Value X F Only in Region Bending DAN Gels 3 Spots 0 Annotations 0 Cancel 7 At any time while the Image Display Settings window is open you can select other gels or redraw the preview regions to see what the effect of the contrast mapping would be on the selected gels 8 Click OK to apply the contrast adjustments to the selected gels Please note that while contrast mapping changes are saved with the gel image without changing the raw data color palettes are not saved in the gel file 2 7 Profile The Profile function gives a horizontal and vertical section of the gel at the position of the mouse cursor thus showing the intensity variation in the X and Y 20 ImageMaster 2D Platinum Tutorial Edition AA Viewing and manipulating gels Kl directions To display the profile ImageMaster automatically loads the raw image data of the gels in the current worksheet 1 Choose Show gt Gels gt Profile in the menu 2 Hold your mouse cursor over the desired gel to view the profiles ul ImageMaster 2D Platinum File view Edit Show Select Analyze Reports Tools Window Help Workspace i Tut
76. s or populations of gels that should be matched together In this tutorial you will match two populations AT1 and AT2 To do so you must first create a match set for each population within the workspace 34 ImageMaster 2D Platinum Tutorial Edition AA Spot detection and gel matching ss 1 In the Gels folder of the Bacteria project expand the folder AT1 2 Select the three gels within AT1 by clicking on their names while holding down the Ctrl key 3 Right click on one of the selected gels and choose Add In MatchSet in the contextual menu 4 Inthe Add Gels In MatchSet window enter AT1 as a name for the match set to be created and click OK 5 Define a reference among the gels which will be used to create a Master image for the matching In our example choose AT1_Gel1 and click OK In the MatchSets folder a match set called AT1 is now displayed which contains the three added gels The gel chosen as the reference has a specific icon with a red component Workspace El Tutorial 1 projects Name MatchSet Ei Bacteria E A_T1_Gel3 mel ati E At1_celz mel ati R AT Gelme Rai cena an 4_T1_Gel2 mel A TI Gell mel Ca AT2 A_T2_Gel3 mel A amp _T2_Gel2 mel B 4_T2_Gell mel MatchSet KN ets a pl 6 Right click on the match set AT1 and choose Open in the contextual menu 7 AlMatchSet worksheet is opened The master gel Master_AT1 is automatically shown with a red legend in this worksheet The reference _ SSSSa
77. spots in the displayed images and those in the Master by choosing Show gt Matches gt Show Vectors in the menu Vectors are shown for all images but the master image These vectors are automatically minimized in the displayed region If the matching results are not satisfying you can add additional landmarks or manual matches In order to optimize the matching avoid placing landmarks in distorded regions Then re run the matching process if you manually added matches keep the existing matches when asked for it You can also check the matches for individual spots using Select gt Matches gt For Spots in the menu This option selects all the matches corresponding to the spots currently selected by the user ImageMaster 2D Platinum Tutorial Edition AA D ImageMaster 2D Platinum Eile View Edit Show Select Analyze Reports Tools Window Help Workspace Project DIGE Ei Tutorial 1 projects Si DIGE Gels Ki DIGE Gels a Gel OI d Gel 02 a Gel 03 a Gel 04 Ca MatchSets SRON DIGE aa Gel 01 Ge Gel 02 ze Gel 03 oR Gel 04 Ga Classes Lei Reports Lei Documents 5 9 Creating classes with DIGE images To find significant protein expression variations between the control and treated samples classes must be created for each type of sample To create a class for the Control samples 1 Inthe Workspace window expand the MatchSets Gel 01 Gel 02 Gel 03 and Gel 04 and select all the images correspon
78. ster 2D Platinum Tutorial Edition AA 39 Spot detection and gel matching oO ImageMaster 2D Platinum DER File View Edit Show Select Analyze Reports Tools Window Help Gels 3 Spots 0 Annotations 0 You can check the vector pattern for consistency If there is a mismatch the vector will have a different length and or orientation 15 Show the spots from the master by selecting Show gt Gels gt Transparency gt Show in the menu In the Transparency Settings window choose Master_AT1 as the transparency reference and tick the Spots Overlapped option then click OK 40 ImageMaster 2D Platinum Tutorial Edition AA Spot detection and gel matching E ImageMaster 2D Platinum File view Edit Show Select Analyze Reports Tools Window Help Gels 3 Spots 0 Annotations 0 16 The red spots are always those from the current gel the blue spots those from the specified transparency reference 17 The blue spots are not necessarily well superimposed on the red spots To solve this make sure the Hand tool is still selected and then double click on a particular position on the gel All your gels now have the same zoom factor and are centered at the same position The distances between the red spots and the corresponding blue spots in the transparency reference are minimized in the clicked region 18 Hide the spots from the master transparency reference by selecting Show gt Gels gt Transparency gt Hide in the me
79. t can be used to close reports ImageMaster 2D Platinum Tutorial Edition AA Experiment setup D ImageMaster 2D Platinum File view Edit Show Select Analyze Reports Tools Window Help Workspace Dy bed Project None o ImageMaster D projects Gels o Spots o Annotations o To revert to the Pinned mode 6 Drag the window title bar in one of the blue guides The window is now in Pinned mode 1 3 Create a new workspace To create a new workspace 1 Click the Workspace tab below the ImageMaster toolbar to display the Workspace window Workspace Project None o ImageMaster O projects ImageMaster 2D Platinum Tutorial Edition AA Experiment setup ss 2 Click on the New icon bh in the workspace toolbar 3 Specify a workspace name and a destination folder for the new workspace file mws You can also enter a comment to help you remember later on what you did and why If you work on the tutorial gels enter Tutorial in the Workspace Name field and browse the folder C imageMaster 2D Platinum Tutorials Tutorial1 to save your file Click OK when you are finished Create New Workspace Enter New Workspace properties Workspace Name Tutoriall Location ImageMaster 2D Platinum utorialsNT utoriall Si Comment SEH LA Add new project In the Workspace window you will find that your new workspace does not contain a project A project includes all gels along with related data produced an
80. t the right side of your screen e Open If you already worked on the tutorial gels saved the changes and closed the images to look at another tutorial or process your own image files this opens the tutorial files with your prior modifications so that you can continue working with the gels and workspace The tutorial instructions are displayed in a browser window that opens up at the right side of your screen If the corresponding tutorial was never loaded before this option automatically restores the initial state of the tutorial e Results Opens the workspace with processed files that can be expected once all the instructions in the corresponding tutorial are run Please note that this overwrites your own work on the tutorial files The tutorial guidelines are displayed in a browser window that opens up at the right side of your screen ImageMaster 2D Platinum Tutorial Edition AA 1 Experiment setup 1 1 Introduction The workspace is the command center of ImageMaster 2D Platinum It can be seen as the place where all gel matching and analysis data is centralized and from where all operations carried out in the software are controlled The workspace is a systematic and efficient way to clarify your analysis and avoid unnecessary work Ideally it should reflect the structure and design of your research It is necessary to setup a workspace to analyze new gels Note that this tutorial in particular describes the processing of non DIGE
81. y3 Control mel Gel 01 Cy5 Treated mel Cancel 68 ImageMaster 2D Platinum Tutorial Edition AA DIGE analysis Kl 8 The DIGE gel Gel 01 appears in the DIGE Gels folder in the workspace project When expanding the gel by clicking on the sign the three DIGE images become visible 9 Repeat the procedure steps 1 to 7 for the Gel 02 Gel 03 and Gel 04 image files 5 4 Co detecting DIGE gels The co detection algorithm is designed to simultaneously process the different images derived from a single DIGE gel To perform spot detection on the four DIGE gels 1 Inthe DIGE Gels folder of the project select the four DIGE gels Gel 01 Gel 02 Gel 03 and Gel 04 by clicking on their names while holding the Ctrl key 2 Right click on one of them and choose Detect in the contextual menu 3 The gel images are loaded and opened in a new worksheet 4 Inthe DIGE Spot Detection window enter an estimation of the Number of Spots present on each image For the current tutorial choose 1500 tick the Apply To All option and click OK DIGE Spot Detection Enter the estimated number of spots for DIGE Gel 01 Number of Spots 1500 Previous estimation Iw Apply to all 5 A status window appears showing the progress of the spot detection Depending on your computer resources the co detection process can take some minutes 6 The spots appear on the gel images Save them by choosing File gt Save gt Worksheet in the menu 7 Adjust th
82. y5 Control mel Gel 01 Cy5 Treated mel Gel 02 Cy3 Treated mel f Gel 03 Cy5 Treated mel 9 Gel 04 Cy3 Treated mel Lei Reports Lei Documents Gels 8 Spots D Annotations 0 5 10 Comparing classes with DIGE images The comparison of classes containing DIGE images is very similar to the comparison of classes with non DIGE gels The only significant difference is that the Volume Ratio is generally the most appropriate value to use in your Inter Class Reports because it uses the internal standard 1 Choose Select gt Gels gt All Ctrl A in the menu to select all the gels from the classes Control and Treated Only selected gels will be included in the analysis ImageMaster 2D Platinum Tutorial Edition AA 79 DIGE analysis 80 2 Select all matches by choosing Select gt Matches gt All Ctrl Shift A in the menu The spots matched to spots in the Master become green 3 Choose Select gt Matches gt Refine Selection in the menu Choose the selected spots is gt option and enter 6 with the scrolling bar This allows you to select only the matches that exist on 6 images or more 4 Choose Analyze gt Inter Class gt Report in the menu 5 Select the value type to be used In DIGE applications using an internal standard including this tutorial the Volume Ratio is most appropriate Click OK Inter Class Report Choose the value type to display Intensity amp Intensity Zol Cancel 6

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