demonstration of 2 -de gel analysis procedure virtual proteomics laboratory iit-bombay

Demonstration of 2-DE gel analysis procedure

Virtual proteomics laboratory IIT-Bombay

Part – IHow to analyze the gels?

Work flow of gel analysiswith commercially available software

1.Set up Project

2. Detect spots

3.Addition and deletion of spots

4.Analyse Expression Profiles

5.Present Results

Gel1 - Control Gel2 - Treatment

File Edit View Reports Tools

Gel1

Gel2

Layout of 2-D gel analysis software showing various tools

Spot analysis

Zoom tool

Crop tool

3-D graphical representation

Image overlaying

1. SET UP PROJECT

Create a project for analysis.

2. Import the 2-DE images



ReportCrop gelsDetect spotsMatch gels

Parameters

Smooth

Saliency

Min Area

Auto Preview

Detect Spots

OK Cancel PreviewPreview

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3. Detection of spots



ReportCrop gelsDetect spotsMatch gels

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Gel matching in progress….

Gel Matching

Matching gels is complete!

9 matches have been created to 1 gel

Gel Matching

4. Gel matching

5. Gel image overlaying



Control + Treatment

6. 3-D View of spots



Part – II Results, Analysis and Interpretation

7. Spot analysis table


Spot # Spot ID Intensity Area Volume % Volume Saliency

1 646 4888 2.95 4720.64 0.0470098 167.048

2 645 1522 16.04 9943.34 0.0990194 101.991

3 644 7776 4.62 11639 0.115906 778.631

4 643 2446 6.66 6171.84 0.0614614 228.525

5 642 1884 9.84 8637.28 0.0860131 100.656

6 641 14444 8.61 52545.8 0.52327 1928.37

7 640 3026 11.55 14935.1 0.148729 398.009

8 639 6194 9.07 19118.9 0.190393 667.417

9 638 4906 12.15 21697.2 0.216069 713.884

10 637 2182 17.45 12250.2 0.121992 332.679

8. Scatter plot for expression pattern of healthy Control vs diseased

Cluster AnalysisHierarchical Cluster(HCL) Analysis HCL Tree

Profile Spot Information Control Case

Spot ID : X pI : 5.26 MW : 65.1kDal

Spot ID : Y pI : 5.28 MW : 44.8kDal

Spot album

Control Case Fused

Control Case Fused

3D view

3D view

4.00 4.25 4.50 4.75 5.00 5.25 5.50 5.75 6.00 6.25 6.50 6.75 7.00

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pI index

Mol

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Figure showing Molecular weight & pI

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Spot ID: X pI : 5.28 Mol. weight : 44.8 kDal

Spot ID: Y pI : 5.26 Mol. weight : 65.2 kDal

No.

of s

pots

sho

win

g up

regu

latio

n

Up regulation No. of folds(and above) No.of spots

2 693 474 395 338 21

10 1615 1520 1025 850 375 1

Up regulationx Folds or above

Differential expression : Up regulation

Expression profile: Up regulation(2-2.5 folds up) -11 spots

A B C D E F

G H I J K

Mean normalized value of group-Control(Healthy person)

Mean normalized value of group 2 (P.falciparum diseased person) Ratio =

Down RegulationNo. of folds(and above) No. of spots

2 83

3 48

4 37

5 28

7 13

10 11

15 5

30 2

No.

of s

pots

sho

win

g up

regu

latio

n

Down regulationx Folds or above

Differential expression : Down regulation

Expression profile: Down regulation(9-10 folds down) - 9 spots

A B C D E

F G H I

Mean normalized value of group-Control(Healthy person) Ratio =

Mean normalized value of group 2 (P.falciparum diseased person)

Conclusion

• 2DE gel analysis using software helps to establish differential expression profile.• Gives the user information about up and down regulated protein spots.• Provides approximate pI and Molecular weight information.•The differential expressed spots may be further subjected to mass spectrometry to allow the further characterization.

demonstration of 2 -de gel analysis procedure virtual proteomics laboratory iit-bombay

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