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Demonstration of 2 -DE gel analysis procedure. Virtual proteomics laboratory IIT-Bombay. Part – I How to analyze the gels?. Work flow of gel analysis with commercially available software. 1.Set up Project 2. Detect spots 3.Addition and deletion of spots 4.Analyse Expression Profiles - PowerPoint PPT Presentation
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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
Gel1 - Control Gel2 - Treatment
File Edit View Reports Tools
ReportCrop gelsDetect spotsMatch gels
Parameters
Smooth
Saliency
Min Area
Auto Preview
Detect Spots
OK Cancel PreviewPreview
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3. Detection of spots
Gel1 - Control Gel2 - Treatment
File Edit View Reports Tools
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
Gel1 - Control Gel2 - Treatment
File Edit View Reports Tools
Control + Treatment
6. 3-D View of spots
Gel1 - Control Gel2 - Treatment
File Edit View Reports Tools
Part – II Results, Analysis and Interpretation
7. Spot analysis table
File Edit View Reports Tools
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
ecul
ar w
eigh
t (kD
al)
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.