Demonstration of 2 -DE gel analysis procedure Virtual proteomics laboratory IIT-Bombay.

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Presentation transcript:

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 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 - ControlGel2 - Treatment File Edit View Reports Tools Report Crop gels Detect spots Match gels Parameters Smooth Saliency Min Area Auto Preview Detect Spots OK Cancel Preview Detection of spots

Gel1 - ControlGel2 - Treatment File Edit View Reports Tools Report Crop gels Detect spots Match gels 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 - ControlGel2 - Treatment File Edit View Reports Tools Control + Treatment

6. 3-D View of spots Gel1 - ControlGel2 - Treatment File Edit View Reports Tools

Part – II Results, Analysis and Interpretation

7. Spot analysis table File Edit View Reports Tools Spot #Spot IDIntensityAreaVolume% VolumeSaliency

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

Cluster Analysis Hierarchical Cluster(HCL) AnalysisHCL 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 3D view

pI index Molecular weight (kDal) Figure showing Molecular weight & pI 85 Spot ID: X pI : 5.28 Mol. weight : 44.8 kDal Spot ID: Y pI : 5.26 Mol. weight : 65.2 kDal

No. of spots showing up regulation Up regulation No. of folds (and above)No.of spots Up regulation x Folds or above Differential expression : Up regulation

Expression profile: Up regulation(2-2.5 folds up) -11 spots ABCDEF GHIJK Mean normalized value of group-Control(Healthy person) Mean normalized value of group 2 (P.falciparum diseased person) Ratio =

Down Regulation No. of folds (and above)No. of spots No. of spots showing up regulation Down regulation x Folds or above Differential expression : Down regulation

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