Transcript appendix 2
Application of 2D in global profiling of E.coli Proteome the Two dimensional electrophoresis can be used top study the entire protein present in the cell which can be used to identify the specific function of each protein Related LOs: Protein property > Prior Viewing – IDD-1. Extraction of bacterial protein, IDD-11. Protein quantification, IDD-14. Isoelectric focusing, IDD-17. SDS-PAGE , IDD-19. Coomassie staining, IDD-23. DIGE gel scanning > Future Viewing – IDD-33. Western blot assay, IDD-35. Immunohistochemistry Course Name: Application of 2D in global profiling of E.coli proteome Level(UG/PG): PG Author(s): Dinesh Raghu, Vinayak Pachapur Mentor: Dr. Sanjeeva Srivastava 1 2 3 Learning objectives After interacting with this learning object, the learner will be able to: 1. Define to design a good experimental plan 2. Identify the significance of 2D separation technique 3. 4. 4 5 Infer the function of hypothetical proteins and the experimental proteins in the study Assess the troubleshooting steps involved in the experiments. 1 Master Layout 2 3 Slide 4-5 4 5 Slide 6-11 1 Step 1: T1: First and second dimension separations Description of the action 2 3 4 5 Instruct the user to go through IDD-1. Extraction of bacterial protein, IDD11. Protein quantification, IDD-14. Isoelectric focusing, IDD-17. SDSPAGE , IDD-19. Coomassie staining, IDD-23. DIGE gel scanning, IDD-24. DIGE gel analysis. Use the pictures from previous slide to show in short the animation for all the above mentioned IDD must be a short recap of the experiment flow. Animate to start with Extraction followed by Quantification, loading of the protein sample on the strip, carrying out the focusing followed by 2-dimensional separation, scanning, staining followed by the gel analysis. Use the pictures from previous slide Audio Narration How the proteome profile of a particular sample starts from Extraction followed by Quantification, loading of the protein sample on the strip, carrying out the focusing followed by 2-dimensional separation, scanning, staining followed by the gel analysis. 1 Step 1: T1: First and second dimension separations 2 3 4 5 The normal gel must be stained later rounded for each protein spot Later protein name annotation of each stop. The protein spot must be pop-out when user goes through the spots. 1 Step 2: T2: Mass spectrometry analysis Description of the action 2 3 4 5 Instruct the user to go through the IIDD-26. Spot picking, IDD-27. In gel digestion, IDD-30. Matrix Instrumentation and IDD-31. MALDITOF data analysis, IDD-39. LCMSMS data analysis. Instruct user to animate the screen with the figures from IDD mentioned with figure options as in next slide. Instruct the user to click on start and show like the processing sign in the screen followed with the screen from the slide:9-12. Wherever “human serum “ in the IDD is found replace all with the “E.coli” for this animation Audio Narration The next part of the Proteome profiling is the identification of the protein after carrying out 1D and 2D separation. The identification part plays a major role, with more improvements in MS instrument as helped proteome analysis at rapid phase. 1 2 3 4 5 Step 1: T1: Title of the step, to appear as heading of the screen (if any) 1 2 3 4 5 Step 2: T2: Mass spectrometry analysis 1 2 3 4 5 Step 2: T2: Mass spectrometry analysis 1 2 3 4 5 Step 2: T2: Mass spectrometry analysis 1 2 3 4 5 Step 2: T2: Mass spectrometry analysis Slide 4,5 Tab 01 Slide 6,11 Tab 02 Tab 03 Tab 04 Tab 05 Tab 06 Tab 07 Name of the section/stage Animation area Interaction 1: slide-6: provide user the proteome profile and let user interrupt the result. Instruction: user must be able to find the higher /low abundance proteins, calculate their pI and molecular weight from the given profile. Interactivity area Button 01 Button 02 Button 03 Instructions/ Working area Credits APPENDIX 1 Questionnaire: Question 1: Global proteome means a) Entire protein content of the cell b) Specific proteins in the cell c) Only metabolically functional proteins d) Transcription factors Question 2 E.Coli is a a) b) c) d) Gram positive bacteria Gram neutral bacteria Gram negative bacteria Mycobacteria Question 3: Proteins are identified by a) Mass spectrometry b) UV –visible spectrometry c) NMR d) IR spectroscopy APPENDIX 2 Links for further reading http://www.matrixscience.com/search_form_select.html 1. Henzel.W.J., Watanabe.C., Stults.J.T. (2003). Protein Identification: The Origins of Peptide Mass fingerprinting. J Am Soc Mass Spectrom, 14(9), pp:931-42. 2. Nesvizhskii , A.I., Vitek, O., Aebersold, R. (2007). Analysis and validation of proteomic data generated by tandem mass spectrometry. Nat.Methods., 49 (1), pp.787-97. 3. Deutsch, E.W., Lam, H., Abersold, R. (2008) Data analysis and bioinformatics tools for tandem mass spectrometry in proteomics. Physiol Genomics. 33 (1), pp:18-25. 4. Yates, JR., 2008. Mass Spectrometry and the Age of Proteome. J.Mass.Spec., 33(1), pp.1-19. Books: Proteomics: A cold spring harbor laboratory course manual by Andrew J L and Joshua L, 2009. APPENDIX 3 Summary The application of 2D involves the global profiling of the b proteome of the organism that can used to correlate its state by validating its protein content and function of protein by 2D.