2 D Electrophoresis

ImageMaster Analysis Software

Contact: Hyuk-Kyu Seoh
Rm (lab): PSC 537

Rm (office): PSC 521
Tel: (404) 413-5379

Location: PSC 537

2D electrophoresis manuals:
Sample Prep
2D Cleanup
2D Quant

GE Amersham Manuals:
Amersham 2-D electrophoresis Manual
Ettan DIGE System User Manual

General Warnings:
Every time you use the 2-D equipment, you MUST sign the logbook.

Image Master 2D Platinum 6.0 Cheat Sheet
This is the Cheat Sheet done by GE/Amersham.

I. Importing Images
A. Open Image Master.
B. Open workspace with a single left click of mouse.
C. Left mouse click on the New Icon to create a new workspace

1. Give it a name and a description (Tutorial 1)
D. Right mouse click on the name of the new workspace – New Project – give it a name (Bacteria).
1. Note a bunch of folders appear.
2. Right mouse click on the Gels folder (Not DIGE).
3. Import Gels – File format is TIFF, Reduction Factor 1.
(Note, if you have converted these gels to .mel files then click Add Gels)
a. Find the gels you want to add, in this case Tutorial – Tutorial 1.
b. Select all of the gels and then select the staining method.
c. Note that the gel now appear in the project.
E. Right Mouse click the Gels folder and Create Folder.
1. Name it AT1 – OK.
2. Select all of the gels with the AT1 name and drag them to that folder.
3. Do the same thing with the AT2.
4. Save.
II. Viewing and Manipulating gels – (Do this several times to get used to it).
A. Display the gels in a worksheet.
1. Open IMPt and click on workspace – the Bacteria from Tutorial 1 should be there, if not open it.
2. Hold down the Shift key and highlight AT1 and AT2.
a. Right mouse click – Open – In worksheet.
3. Click on the worksheet tab and then the pin in the upper right hand corner.
4. Click on the AT1 pane and the AT2 becomes gray (Don’t do it but Shift – click on AT2 turns all of them green again).
5. File – Close Images closes the AT1 pane and the AT2 is left.
a. Click on the AT2 pane to turn it green.
6. In the workspace, right mouse click on the AT1 folder – Open – In New Worksheet.
7. Note that you now have two gel sets AT1 and AT1and AT2.
a. Clicking on the gray tab of the pane makes it the active one.
8. Highlight the AT1 and AT2 [Gels] pane and then File – Close Images.
a. Only the AT1 [Gels] pane should be open.
B. Handling the gels.
1. Click on the hand tool icon at the top left.
a. Move it to the gel 1 image and hold down the left mouse while you move it around.
b. Note what happens to the image as well as the slide bar below the image.
c. Shift double left mouse click moves all of the other images to the same spot.
1). If you held the shift key it would have done it immediately.
2. Click on the magnifying lens at the top left.
a. Go to image 1 and single left mouse click to magnify the image.
1). Shift single mouse left click makes all images the same size.
2). Holding the Shift key when you click on am image will adjust all of them immediately.
b. Right mouse click reduces the image size.
3. Click on the dotted box at the top left.
a. Go to image one and Hold down the left mouse – draw a box around the region of interest.
b. Shift key does what it did before.
c. Move the mouse outside the box you drew and Shift double left click removes the box.
d. Make sure the AT1 pane is clicked so all three gels are green before the next step.
C. Organize the Gels.
1. View – Pane layout – Stacked.
a. Images are now layered one upon the other.
2. Clicking on the green banner at the bottom left brings that image to the front – Page up, Page down does the same thing.
3. If all of the gels are not in the same position, click on the hand tool at the top left and then double click on the image to center all of the images.
4. View – Pane Layout – Free Select 3 columns and 1 row.
5. View – Pane Layout – Tiled goes back.
D. Adjust contrast.
1. Draw a region of interest around one or more of the gels – Shift key does all the gels.
2. Show – Gels – Adjust Contrast.
3. Move the slide bar (left mouse), and play with the bending.
a. Note what happens in the box you drew.
b. Get the best you can with the two.
4. There is also a color drop-down box – Play with the colors.
5. Click OK and it applies it to the entire image(s).
6. Remove the region of interest box.
E. Profile.
1. Show – Gels – Profile.
a. Move the mouse around and notice what happens.
b. Show – Gels – Profile removes it.
2. View – Pane Layout – Free 1 column, 3 rows and then go on to next section.
F. 3D View
1. Draw a region of interest around one of the Gels.
2. Reports – 3D View.
3. Left mouse click on the pin in the upper right of the 3D view to pin it next to the image.
a. In the 3D view box is a globe with arrows on it: - hold the left mouse on one of the arrows to rotate the 3D view.
4. Using the Shift key before you do the region of interest will give the 3D view for all of the images.
5. Play with this and then close the 3D window.
G. Cursor information.
1. Windows – Cursor Information.
a. Move the mouse around the gel and see what happens.
2. Click on the Settings box of the Cursor Information window (upper left).
a. You can move things and hide them if you want.
3. Close the cursor information box.
III. Spot detection and Gel Matching

A. Open gels and Displaying spots
1. Select subfolders AT1 and AT2 – right mouse click – Open In Worksheet.
a. Hide the Workspace window.
2. Select all gels – Ctrl A
a. Show – Spots – Shape – Outlined.
b. Since no spots are detected yet you really won’t see anything.
B. Spot Detection – Select all gels with Ctrl A.
1. Draw a region of interest around one of the gels including some good spots.
2. Edit – Spots – Detect.
a. Adjust the Smooth parameter – Play with this to see what it does then set it to 2 for the Tutorial 1 gels.
b. Window – Cursor Information – Move the cursor over some good spots and see what the Saliency is. Then move it over some outlines that really don’t contain protein.
c. Set the Saliency for 150 for the tutorial.
d. Click Ok.
3. File – Save – Worksheet.
4. Select the Region tool and make the region go away.
C. Editing Spots. – They aren’t big on editing.
D. Spot Report.

1. Click on the Spot tool (upper left)
2. Click on about three or four spots on one of the gels using the shift key
3. Reports – Spot Report –Current Template – gives you a table.
a. Click on the Settings icon (2nd from right) in the table.
b. You can choose the information you want to add to the table.
c. You can print it, export it to Xcel etc.
4. Close the Report window.
5. File – Close All. This closes the AT1_AT2 worksheet.
E. Creating Match Sets
1. Open the Program and Expand the AT1 folder.
2. Select all of the gels in the folder and right mouse click – Add in MatchSet. Type in AT1 then OK.
a. Define a reference: use AT1_Gel1 – OK.
b. If you open the AT1 MatchSet you can see that Gel 1 has a different icon
3. Right mouse on MatchSet AT1 – open.
a. Show – Spots – Shape – Outlined if needed.
4. Do steps 1-3 above for AT2.
5. Right mouse on the MatchSet folder and choose Create MatchSet
a. Enter A as the new match set and then OK.
6. Drag and drop the match sets AT1 and AT2 into the new A folder.
7. A pop-up appears that asks if you want to move them inside or after A. Check Inside.
a. The match sets AT1 and AT2 should now be in match set A.
8. Right mouse on match set AT1 and choose Set MatchSet as Reference – It will now have a different Icon.
F. Matching Gels
1. Right mouse on match set AT1 – Open.
2. Ctrl A – Select – Unselect All.
3. Click on the spot icon and then Shift – Select the same spot on all of the images.
a. It should be small, circular and well defined.
4. Edit – Annotations – Add Label.
5. Choose Landmark – OK
6. Supply name L1 – OK – close the window.
7. Perform matching.
a. Ctrl A.
b. Edit – Matches – Match Gels.
c. OK.
d. File – Save – Worksheet.

8. View matching results.
a. Show – Matches – Show Vectors. – This shows you how good of a job it did matching.
b. Show – Gels – Transparency – Show – Choose Master_AT1as reference – Click Overlapped. – OK.
c. Click on the hand tool and then click on a spot. The others will line up.
d. Show – Gels – Transparency – Hide – hides the spots from the master.
9. Editing.
a. Click on spot tool – click on the same spot in all three images – Edit – Matches – Delete Match to remove matches.
b. Edit – Matches Add Match to add matches
c. Shift – Edit – Matches – Add Match to add matches to all selected spots.
10. Repeat the procedure to do Match Set AT2.
IV. Data Analysis – Ok let’s keep it simple and still work on Tutorial I and then we can jump to Tutorial 4.
A. Right mouse Match Set folder- Create Match Set – Label it Abis.
1. In the Gels folder open the AT1 and the AT2 folders. Highlight all six gels and drag them to the new Abis folder. (This is done so all gels are matched to each other for comparison in Classes)
2. Right mouse the Abis folder and Open.
a. Show – Spots – Shape –Outlined.
b. Find the annotations, select the spot tool and using the shift key click on all of them – Edit – Annotations – Delete.
3. Using the spot tool, and Shift key click on a spots common to all six gels.
a. Edit – Annotations – Add Label – Landmark – L1.
4. Edit – Matches – Match Gels.
a. Check matches by Show – Gels – Transparency – Show – Spots Overlayed.
5. Save – Worksheet.
B. Create classes
1. Click on the all of the AT1 gels in the Abis Match set – Right mouse click – Add In Class – call it AT1
2. Repeat for AT2 gels.
3. Click on AT1 and AT2 in the Classes folder – Right Mouse – Open.
4. Ctrl A.
5. Show – Spots – Shape – Outlined
6. Select – Matches – All. – Some of the rows turn colors
C. Create Reports.
1. Analyze – Iner-Class – Report - %Vol – OK – OK.
2. Change Center to Ratio in the Inter-class Reports window.
3. Click on the Save icon in the Inter-Class Reports window and save the report:
4. In the report window choose the Select on Gels icon – (it’s the 5th from the left).
a. In the dropdown choose Refine Selection
b. Choose Max – OK.
c. Make sure the upper box is checked and enter 2.0 in the space – uncheck the lower box.
d. Some of the rows are now in green.
5. In the Reports icon (10 from left) select Report from Selection.
6. In the Report Window choose the far right box – it says Ratio.
a. Select Gap from the dropdown.
7. In the Main view select Window – Ratio Report and close it – only the gap report is now open.
a. Click the Max column once to arrange the values
8. In the Report toolbar click on the Annotate icon – (8 from the left).
a. Click on Set- then type in the box nest to Set Verified – OK – OK – note the extra column in the report.
9. Click on the box to the left of Match ID in the report and all of the boxes turn green.
10. Click on the Histogram icon on the Report toolbar – (11 from left) – Choose Inter-Class+ Intra-Class Histograms option. – Pin it.
11. Now select the first line in the report.
a. Double clicking it makes the spots appear.
b. View the histogram with the same number
c. If you like it check the Set:Verified box.
d. Do this for all of them in the report.
e. Remember you can use the 3D view to check spots too.
11. Click on the box to the left of the ID Match column header – all of the rows should turn green.
12. Click on the Update gels icon in the Report section (9 from left).
a. Answer Yes.
13. Ctrl A to select all gels
14. Edit – Annotations – Copy Matched labels.

Let’s Do it Start to Finish

I. Discussion.
A. There are several steps that must be conducted in the following order.
1. Open Images
a. Adjust contrast.
b. Can look at Profile.
c. Can look at 3D view.
d. Can look at Cursor Information.
2. Spot Detection
a. Adjust Smoothing.
b. Adjust Saliency.
3. Create Match
a. Put all of the images you will want to compare into the same Match Set folder.
b. Show spots in Outlined Mode.
c. Add one clearly defined reference landmark on all of the images
d. Match gels.
e. Check matches with the vectors and transparency.
4. Create Classes.
a. Create a Class for all gels that are the same.
b. Put the gels in the Class folder.
5. Create Reports.
a. Create an Inter Class report from the Classes
b. Make the report into a ratio report.
c. Refine the report.
d. Using the histogram and 3-D views select the spots of interest.
e. Transfer the spots to all of the images.
f. Save.
II. Analysis.
A. Open Images and Create a New Workspace - Final.
1. Open Workspace and Create a New Project – Start to Finish.
2. Right click on the New Workspace and Create a New Project – Test.
3. Right Click on the Gels folder – Import Gels – TIFF – Tutorials – Tutorial 1.
a. Highlight all of the Images – Open – Yes – Choose the stain (Coomassie).
4. Right click on the first gel (They should all be highlighted) –Open – In Worksheet.
5. Adjust contrast.
a. Draw a box around all of the gels with the shift key.
b. Show – Gels – Adjust contrast.
1). Move slide bar.
2). Adjust Bending
(can view Profile and 3D if you want too).
B. Spot Detection.
1. Show – Spots – Shape – Outlined.
2. Ctrl A.
3. Draw a region of Interest around one of the gels.
4. Edit – Spots – Detect.
a. Adjust Smoothing (2)
b. Window – Cursor – Cursor Information – move across spots to determine Saliency.
c. Adjust Saliency (150).
d. OK
e. File – Save – Worksheet.
f. File – Close Images.
C. Create Match.
1. Right Mouse on MatchSet folder.
2. Create MatchSet – A Samples.
3. Drag all of the Gels from Detection into the A Samples folder.
4. Select the A1 Gel as the Reference.
5. Right Mouse – Open.
6. Show – Spots – Shape – Outlined.
7. Draw the same Region of Interest around all of the gels.
8. Select spot icon and shift key to select the same spot on all the gels.
a. Edit – Annotations – Add Label – Landmark – OK.
1). Type L1 – OK – Yes.
b. With the region tool icon selected use the shift key and double click outside of the box.
9. Edit – Matches – Match Gels – OK.
a. Show – Gels – Transparency – Show – Spots Overlayed to check.
b. Check Vectors too – Show Matches – Show Vectors.
10. File – Save Worksheet.
11. File – Close Images.
D. Create Classes.
1. Right Mouse on the Classes folder and create two separate classes, AT1 and AT2.
2. Drag the AT1 Images into the AT1 folder and the AT2 Images into the AT2 folder.
3. Highlight the two new folders and open.
4. Show – Spots – Shape – Outlined.
5. Select – Matches – All.
E. Reports.
1. Analyze – Inter-Class – Reports – %Vol – OK – OK. Pin the window.
2. Change Center to Ratio in drop down of Report.
a. Save the Ratio Report – Give it a name – Ratio
3. Click the Select on Gels icon (5 from left) and choose Refine Selection – Max – OK.
a. Type 2 in the upper and unclick lower – OK.
4. Reports icon (10 from left) – Report from Selection.
a. Change the Ratio to Gap in the drop down.
b. Window – Choose the Ratio report and close it.
5. Click the Max column in the new Report.
6. Click the Annotate icon (8 from left) – Set Type in Verified next to the set up top – OK – OK.
7. Click the Box to the left of Match ID – all turn green.
8. Click the Histograms icon (11from left) – Inter-Class + Intra-Class Histograms. – Pin it.
9. Select on gels icon (5 from left) – Select on Gels + Reports.
10. Double click on the first row – check the gels and the histogram.
a. If you like it click the Set Verified box – You can use the 3D view too.
b. Do the same for all of the rows.
11. Click on the box to the left of Match ID to turn all rows green.
12. Click on the Update Gels icon (9 from left) – Yes
13. Ctrl A
14. Edit – Annotations – Copy Matched Labels. –Save
V. DIGE Analysis.
A. Open Workspace – New – Name it Tutorial 5.
1. Right mouse – New Project – DIGE.
B. Import Gels.
1. Right mouse on DIGE Folder
2. Import DIGE Gel
a. Reduction 1
b. File format – Gel.
c. OK
3. Click on Gel 1 and then using shift key highlight all of the Gel 1 images – Open.
a. DIGE Min should be checked
b. Staining – Cy2 – OK.
c. Answer any other questions.
d. Do this for all four gels.
C. Co-Detection.
1. In DIGE Folder, highlight all 4 DIGE gels using shift.
2. Right mouse – Detect
3. Select number of spots (1500 for this tutorial).
4. Also check the apply to all box.
5. Show – Spots – Shape – Outlined.
6. File – Save – Worksheet.
7. Adjust the contrast on the gels.
a. Show – gels – Adjust Contrast.
D. Display DIGE Histogram.
1. Ctrl select Gel 1 Cy 3 control and Gel 1 Cy 5 treated.
2. Select – Spots – All.
3. Analyze – DIGE – Histogram.
a. On Histogram – Measure (4 icon from left) – Max Volume.
b. Use Ctrl and click on several of the spots in the histogram.
c. Select on Gels icon (5 from left) – Select on gels.
d. Spots are now on image.
E. Display a DIGE Report
1. In the Report icon of histogram (last on the right) – Reports – DIGE.
a. Shows reports of spots selected above
b. File – Close Images.
F. Create Match Set
1. In DIGE Folder select all four DIGE gels .
2. Right mouse – Create MatchSet – enter DIGE in window
3. Click ok for each gel
4. Right Mouse on DIGE in MatchSet folder – Open
a. Choose a reference – They recommend 3
5. Show – Spots – Shape – Outlined.
G. Match Gels
1. Ctrl A then Select – Unselect All (it may be grayed out –it’s ok)
2. Use the spot icon and shift key to select the same spot in all four images.
a. Edit – Annotations – Add Label – Landmark – OK
b. Call it L1
3. Ctrl A
a. Edit – Matches – Match Gels.
b. OK at end
c. File – Save Worksheet.
4. View Matches.
a. Show – Matches – Show Vectors.
b. Show – Gels – Transparency – Show – Spots Overlayed.
c. File – Close Images.
H. Create Classes.
1. Expand all of the gels in the MatchSet window.
2. Ctrl – highlight all of the Control images
3. Right mouse - Add in Class – type Control – Ok
4. Do the same for treated.
5. Use the Ctrl and highlight the control and treated classes – Right mouse – open.
6. Show – Spots – Shape – Outlined.
I. Comparing.
1. Ctrl A to select all gels
2. Ctrl + Shift + A or Select – Matches – All.
3. Select – Matches – Refine Selection.
a. Choose selected spots is > and enter 6 – Ok (only spots in 6 or more images will be included).
4. Analyze – Interclass – Report.
a. Vol ratio – Ok.
b. Mean 100, MSD 100%
5. Select Ratio from displayed value in report
a. Save icon in report and save report as Ratio in tutorial 5 folder.
6. Click on Select on Gels icon in report (5 from left).
a. Refine Selection – Max Ratio – higher than 2. Make sure bottom box is not checked – OK
7. In the Reports drop down select Gap.
a. Window – find the ratio report and close it. This leaves the Gap open.
8. Click the Max column once in the report to arrange values.
9. Annotate icon (8 from left) – Set type Verified next to set up top.
a. A new column appears.
10. Click on box left of Match ID column to highlight all.
11. Histogram icon ( 11 from left) – Inter-class + Intra-class Histograms. – dock it.
12. Select first line of report.
a. Should see the gel spot.
b. find the corresponding number in the histogram report.
13. Click the Set:Verified box if you like it.
14. Can also do the 3D view: Window – Mouse Selection – 3D view.
15. Select all rows by clicking the box next to Match ID.
16. Click on the update icon (9 from left).
a. Answer yes to add label to one gel (Master).
17. Click on the save icon in Report and save it as Gap in tutorial 5 folder.
18. File – Save – Save All.
J. Matching to a Prep Gel.
1. Right mouse on the DIGE Gel folder.
2. Import – DIGE Gel.
a. Reduction Factor 1
b. Format – Gel – OK
3. Browse Tutorial 5 and select Pick gel
4. OK – enter name Pick.
a. Choose Sypro Ruby – OK.
5. Right Mouse on the Pick Gel
a. Detect – enter 1500 for spots – OK.
b. File – Save Worksheet. Close images
6. Right mouse Pick gel and drag it to the MatchSet DIGE folder.
7. Right mouse Ctrl Pick and Master gel in MatchSet folder – Open.
8. Edit – Matches – Match Gels.
a. Pick is matched to the Master
b. Ok when done
c. Check matches
K. Pick List
1. Select – Annotations – by Category – Set:Verified
2. Select – Matches – For Spots
3. Unselect Master Image by clicking on the legend tab and holding the Ctrl Key.
4. Edit – Annotation – Add Label.
a. Set – type in Pick - OK – OK.
5. Reference Markers.
a. Select Annotation tool (5 from right)
b. Double click on the left reference Marker
c. Select the Comment category – OK.
d. Enter IR1 – OK
e. Do the same for the right reference marker.
f. Make sure only the pick gel is selected.
g. Select – Annotations – By Category – choose Set:Pick – OK.
h. File – Export – Spots to Spot picker – GE Healthcare Ettan.
i. Give it a name



This information is given as a guide to the facilities and instrumentation available in the DNA/Protein Core facility at Georgia State University. If you have any concerns or thoughts about the content of this website please contact: John Houghton (404) 413-5390



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