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

2. Part – I How to analyze the gels?

3. 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

4. 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

5. 1. SET UP PROJECT Create a project for analysis.

6. 2. Import the 2-DE images

7. 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 60 9 4 1 2 3 4 5 6 7 8 9 10 1 2 3 4 5 6 7 8 9 3. Detection of spots

8. Gel1 - ControlGel2 - Treatment File Edit View Reports Tools Report Crop gels Detect spots Match gels 1 2 3 4 5 6 7 8 9 10 1 2 3 4 5 6 7 8 9 Gel matching in progress…. Gel Matching Matching gels is complete! 9 matches have been created to 1 gel Gel Matching 4. Gel matching

9. 5. Gel image overlaying Gel1 - ControlGel2 - Treatment File Edit View Reports Tools Control + Treatment

10. 6. 3-D View of spots Gel1 - ControlGel2 - Treatment File Edit View Reports Tools

11. Part – II Results, Analysis and Interpretation

12. 7. Spot analysis table File Edit View Reports Tools Spot #Spot IDIntensityAreaVolume% VolumeSaliency 164648882.954720.640.0470098167.048 2645152216.049943.340.0990194101.991 364477764.62116390.115906778.631 464324466.666171.840.0614614228.525 564218849.848637.280.0860131100.656 6641144448.6152545.80.523271928.37 7640302611.5514935.10.148729398.009 863961949.0719118.90.190393667.417 9638490612.1521697.20.216069713.884 10637218217.4512250.20.121992332.679

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

14. Cluster Analysis Hierarchical Cluster(HCL) AnalysisHCL Tree

15. 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

16. 4.004.254.504.755.005.255.505.756.006.256.506.757.00 80 75 70 65 60 55 50 45 40 35 30 25 20 15 10 5 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

17. No. of spots showing up regulation Up regulation No. of folds (and above)No.of spots 269 347 439 533 821 1016 15 2010 258 503 751 Up regulation x Folds or above Differential expression : Up regulation

18. 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 =

19. Down Regulation No. of folds (and above)No. of spots 283 348 437 528 713 1011 155 302 No. of spots showing up regulation Down regulation x Folds or above Differential expression : Down regulation

20. 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)

21. 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.