1. 2-DE gel analysis Harini Chandra
Following 2-D gel electrophoresis, the gels are scanned using a suitable scanner and the images obtained are analyzed by means of various available softwares. These allow interpretation of data present in the form of spots on the gel images.
Title of the concept
Harini Chandra

2. 1 Master Layout 2 3 4 5 2-D gel analysis software Zoom tool
3-D graphical representation
2-D gel analysis software
Spot analysis
Image overlaying
File
Edit
View
Reports
Tools
Crop tool 2
Gel1
Gel1 - Control
Gel2 - Treatment
Gel2 3 4 5

3. Definitions of the components:1
1. 2-D gel analysis software: Following 2-D electrophoresis, the gels obtained are scanned using a suitable scanner and the images are then analyzed using various software packages. These softwares enable spot identification, comparison of gels, overlaying of images, cropping the gels as well as computation of more advanced statistical parameters. These help in interpreting the gel images obtained.
2. Crop tool: This tool allows a specific defined region of the gel to be cut out from the entire gel. This helps in selection of regions that have high spot density for further analysis.
3. Zoom tool: The zoom tool enables a specific area or spot of the gel to be zoomed into for further careful examination and analysis.
4. Image overlaying: In order to be able to effectively compare the spot patterns on two different gels, overlaying of the two separate images is possible. The two gels are overlaid such that they appear as a single merged image. Spots that coincide lie on top of each other while others retain their original position on the gel.
5. 3-D graphical representation: Spot patterns obtained can be plotted as three dimensional graphs which are indicative of their intensity. Either the entire gel can be represented as a 3-D graph or a certain region can be selected for representation.
6. Spot analysis: It is possible to obtain physical as well as statistical parameters for each of the spots on the gels. This allows gels to be easily compared on a spot-by-spot basis thereby facilitating the process of analysis. 2 3 4 5

4. Step 1: Loading a gel image
File
Edit
View
Reports
Tools
New
Load
Save
Save as…
Print
Exit
Gel 1
Gel 2
Gel 3
Gel1 - Control
Gel2 - Treatment 2 3 4
Action
Description of the action
Audio Narration
First sow the entire software features zooming in. Mouse pointer must click on ‘file’, then move to ‘load’ and click on ‘gel1 ‘ and then ‘gel2’ respectively. When gel1 is clicked, the image on left should appear and when ‘gel2’ is clicked, image on the right must appear.
It is possible to load either a single or multiple gel images simultaneously. This is done by means of the ‘load’ option in the file menu. The saved gel images must be chosen, which are then displayed on the software.
As shown in the animation. 5

5. 3 1 2 4 5 Step 2: Image cropping Action Description of the action
File
Edit
View
Reports
Tools
Report
Crop gels
Detect spots
Match gels
Gel1 - Control
Gel2 - Treatment 2 3 4
Action
Description of the action
Audio Narration
Mouse pointer must click on the second image tab having the rectangle on it. The dotted rectangles on the gel images must appear. Pointer must then click on ‘edit’ followed by ‘crop gels’ and the figures below must appear.
Several tools are available for analysis of the gels. It is possible to crop the gels by selecting a specific region that is to be studied and then selecting the ‘crop gels’ function. Cropping gels helps in selection of regions with high spot density or in trimming of regions with high background staining but no spots.
As shown in the animation. 5

6. Step 3: Zooming into a selected region1
File
Edit
View
Reports
Tools
Gel1 - Control
Gel2 - Treatment 2 3 4
Action
Description of the action
Audio Narration
Mouse pointer must first click on the second picture tab. The dotted rectangles must then appear. It must then move to the next tab with magnifying glass and click on that. When this happens, the boxed region must be zoomed into to show images below.
Specific, selected regions of the gel can be zoomed into for viewing the spots more closely and for comparison of spots between two gels. This is particularly useful for gels having large number of spots.
As shown in the animation. 5

7. Step 4: Image overlaying1
File
Edit
View
Reports
Tools
Gel1 - Control
Gel2 - Treatment 2
Control + Treatment 3 4
Action
Description of the action
Audio Narration
Mouse pointer must click on the last picture tab having the two overlapping rectangles. When this happens, the two gels must merge into one another to give the gel image in the centre as shown.
Overlaying of images is a particularly useful tool for comparison of two gels. The gels are overlaid such that they appear merged and spots that coincide will overlap with each other. This is extremely helpful while comparing clinical samples of control and treatment, providing clear indication of the proteins that are differentially expressed.
As shown in the animation. 5

8. Step 5: Graphical representation1
File
Edit
View
Reports
Tools
Gel1 - Control
Gel2 - Treatment 2 3 4
Action
Description of the action
Audio Narration
Mouse pointer must click on the fifth picture icon containing the graph. When this happens, the two graphical representations below must appear.
The spots on the gels can be displayed as three dimensional graph. Either the entire gel can be chosen or a particular region can be selected for this representation. The peaks obtained in the graphical representation are directly related to the spot intensity.
As shown in the animation. 5

9. 3 1 2 4 5 Step 6: Spot analysis Action Description of the action
File
Edit
View
Reports
Tools
Report
Crop gels
Detect spots
Match gels
Gel1 - Control
Gel2 - Treatment 1 2 3 4 5 6 7 8 9 10 1 2 3 4 5 6 7 8 9 2
Parameters
Smooth
Saliency
Min Area
Auto Preview
Detect Spots 3 9 60 4 OK
Cancel
Preview 4
Action
Description of the action
Audio Narration
Mouse pointer must click on ‘edit’ followed by ‘detect spots’. The popup box shown in the centre must appear. Mouse pointer must drag the slider bars as shown to the required values and then click on ‘OK’. When this is done, the red crosses and numbers on the spots must appear.
Every spot on the gel can be detected by selecting the ‘detect spots’ option. Parameters such as ‘smoothness’, ‘saliency’ and ‘min area’ must be suitably adjusted for maximum clarity. Once this is done, each spot will either be encircled or marked with a cross, depending on the settings, along with the spot numbers.
As shown in the animation. 5

10. 3 1 2 4 5 Step 6: Gel matching Matching gels is complete!
File
Edit
View
Reports
Tools
Report
Crop gels
Detect spots
Match gels
Gel1 - Control
Gel2 - Treatment 1 2 3 4 5 6 7 8 9 10 1 2 3 4 5 6 7 8 9 2 3
Matching gels is complete!
9 matches have been created to 1 gel
Gel matching in progress….
Gel Matching
Gel Matching 4
Action
Description of the action
Audio Narration
Mouse pointer must click on ‘edit’ and then ‘match gels’. The popup must appear saying ‘matching in progress’ followed by the next popup saying ‘matching is complete’. When this happens, the red crosses & numbers must appear along with the blue lines shown in ‘gel2’ image.
The software facilitates interpretation of the gel images by matching two different gel images obtained. The matching spots are marked and any variations in the spot position are indicated by blue lines in one of the gels. This provides an understanding about the reproducibility across gels.
As shown in the animation. 5

11. 3 1 2 4 5 Step 7: Spot table Action Description of the action
File
Edit
View
Reports
Tools
Spot #
Spot ID
Intensity
Area
Volume
% Volume
Saliency 1
646
4888
2.95 2
645
1522
16.04 3
644
7776
4.62
11639 4
643
2446
6.66 5
642
1884
9.84 6
641
14444
8.61 7
640
3026
11.55 8
639
6194
9.07 9
638
4906
12.15 10
637
2182
17.45 2 3 4
Action
Description of the action
Audio Narration
Mouse pointer must click on the sixth pictorial icon showing the table. When this happens, the table must appear.
Information regarding the various physical parameters of each spot can be obtained via the spot table. This table provides the spot number, intensity, area and volume of spots as well as the saliency of spots. These parameters help in judging the quality of a gel.
As shown in the animation. 5

12. Step 8: Statistical gel analysis1
Measure of the variability of sample data around their centre value.
Measure of relative variability of spots in a match, after accounting for magnitude of data values.
File
Edit
View
Reports
Tools
Gives the general location of a variable and is calculated by arithmetic mean, median or midrange.
Ratio of the maximum value to minimum value in the sample data specified.
Gel analysis
Statistics
Spot comparison
Scatter plots
Histogram
Spot #
Match ID
Central tendency
Dispersion
Coeff variation
Range ratio 1 2 3 4 5
3.2711 6 7
0.5585 8 9
1.1758 10 2 3 4
Action
Description of the action
Audio Narration
Mouse pointer must click on ‘reports’ followed by ‘gel analysis’. The table must appear followed by each of the parameters getting highlighted sequentially along with its callout providing the meaning. Next, mouse pointer must click on ‘reports’ and then ‘scatter plot’. This plot shown must popup when this is done.
In addition to physical parameters, several statistical parameters can also be computed for each spot on the gel such as central tendency, mean, median, dispersion, coefficient of variation, standard deviation etc. Scatter plots and histograms can also be plotted for clear data analysis. These provide information regarding intra and inter-gel variations.
As shown in the animation. 5

13. 3 1 2 4 5 Step 9: Spot comparison Action Description of the action
File
Edit
View
Reports
Tools
Gel analysis
Statistics
Spot comparison
Scatter plots
Histogram 3 7 pI 80 40 20
MW (kD)
Gel1 - Control 2
Spot # 5
Parameter
Gel 1
Gel 2 pI
4.8
4.9 MW
43 kD
37 kD
Spot area
5.43
4.88
Volume
Intensity
1576
1843
Central tendency
0.0789
0.0654
MW markers 5 3 pI 7 3 80 40 20
MW (kD)
Gel2 - Treatment 5 4
Action
Description of the action
Audio Narration
Mouse pointer must first click on the second pictorial tab. The dotted black rectangles and the number must appear. Next the mouse must click on ’reports’ and ‘spot comparison’. When this happens, the table on the right must appear.
It is also possible to specifically compare a particular selected spot across gels. When the gel is run with molecular weight markers, the weight of unknown proteins can be estimated from these. It is also possible to estimate the pI of the proteins. These parameters, in addition to other physical & statistical parameters, can be obtained for each spot.
As shown in the animation. 5

14. 3 1 2 4 5 Step 10: DIGE analysis Action Description of the action
File
Edit
View
Reports
Tools
Report
Crop gels
Detect spots
Match gels
Gel1 – Control – Cy3 labeled 2 1 2 3 4 5 6 7 8 9
Gel3 – Internal standard – Cy2 labeled 1 2 3 4 5 6 7 8 9 3
Enter no. of spots to be detected
Detect Spots
Gel2 – Treatment – Cy5 labeled 9 1 2 3 4 5 6 7 8 9 4
Action
Description of the action
Audio Narration
Mouse pointer must click on ‘edit’ and then ‘detect spots’. The popup box shown must appear where the number 9 must be typed as shown. This is followed by appearance of the pink crosses along with the numbers in all the three gels.
Software for DIGE analysis varies with respect to certain features compared to normal 2-DE analysis. It can compare three gels simultaneously, of which one is typically the pooled internal standard containing all spots. Any changes implemented in one gel such as cropping, spot selection etc. will be implemented across all three gels in DIGE.
As shown in the animation. 5

15. 3 1 2 4 5 Step 11: DIGE analysis Action Description of the action
File
Edit
View
Reports
Tools
Gel1 – Control – Cy3 labeled
Other analysis tools for DIGE are the same as those used for regular 2-DE analysis as depicted in the earlier animations. 2 1 2 3 4 5 6 7 8 9
Gel3 – Internal standard – Cy2 labeled 3 1 2 3 4 5 6 7 8 9
Gel2 – Treatment – Cy5 labeled 1 2 3 4 5 6 7 8 9 4
Action
Description of the action
Audio Narration
The five pictorial tabs shown must be highlighted and must then grow in size as shown in animation, after which the text on the right must appear.
Other features and tools for DIGE analysis are same as those used for 2-DE analysis. Physical and statistical parameters of all spots on the gels can be determined through their corresponding reports. Three dimensional graphical representation provides information regarding intensity of spots on the gel.
As shown in the animation. 5

16. Interactivity option 1:Step No: 1 (a)
Find the number of matched spots in the gel below by performing the required steps in the software.
File
Edit
View
Reports
Tools
Report
Crop gels
Detect spots
Match gels 2
Gel1 - Control
Gel2 - Treatment 1 2 3 4 5 7 6 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 1 2 3 4 5 7 6 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 3
Gel Matching
Matching gels is complete!
21 matches have been created to 1 gel
Gel Matching
Gel matching in progress…. 4 5

17. Interactivity option 1:Step No: 1 (b)
Find the number of matched spots in the gel below by performing the required steps in the software.
A) 23 2
B) 21
C) 25 3
D) 24 4
Interacativity Type Options
Results 5
User must be allowed to perform the required steps in the software layout shown in the previous slide. The edit tab must be active for user to click on followed by the ‘match gels’ option. Once the user has clicked on these, the animation must appear as depicted. Once the ‘matching gel is complete’ appears, the question above must be displayed & user must be allowed to choose 1 option.
The correct answer is (B). If user chooses this, then it must turn green. If user chooses any of the other options, they must turn red.
Clicking on tabs &
Choose the correct answer.

18. Questionnaire 1
1. Which of the following tools helps in trimming unwanted regions of the gel?
Answers: a) Zoom b) Match c) Crop d) Overlay
2. Gel A has 20 spots and Gel B has 18 spots, of which 10 are common to both gels. The total number of spots in the overlaid image would have:
Answers: a) 38 spots b) 18 spots c) 30 spots d) 28 spots
3. Which of the following is NOT a statistical parameter?
Answers: a) Spot intensity
b) Dispersion
c) Coefficient of variation
d) Median
Which of the following is not a dye used in DIGE analysis?
Answers: a) Cy-3 b) Coomassie blue c) Cy5 d) Cy2 2 3 4 5

19. Links for further reading
[ImageMaster]