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BIS 104 PQ 20 ans Spring 2010

BIS 104 PQ 20 ans Spring 2010 - 6 Compare the banding...

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BIS 104 PQ 20 ans Spring 2010 20. Let’s suppose that you have recently discovered an extraordinarily simple cell that has only two different proteins associated with its plasma membrane. Preliminary analysis shows that protein “A” has a mol wt of 45,000 Da and protein “B” has a mol wt of 60,000 Da. You hypothesize that protein “A” is an external peripheral protein and that protein “B” is a GPI- anchored protein. Describe an experimental protocol that would test this hypothesis. 1. Divide your healthy, viable cells into three equal batches: a, b,and c. 2. Do not treat batch a. 3. Subject batch b to a gentle ionic wash (no permeabilization). 4. Subject batch c to trypsin treatment (no permeabilization). 5. Run a total membrane extract from each of the batches side-by-side in 3 SDS-PAGE lanes and stain for proteins with Coomassie Blue.
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Unformatted text preview: 6. Compare the banding pattern in the three lanes for extracts a, b, and c. Describe the results you would obtain if your hypothesis were shown to be correct. 1. Lane for batch a would contain two bands: one at 45kDa, one corresponding to 60kDa. 2. Lane for batch b would be missing protein band “A”; band for protein “B” would not be altered. 3. Lane for batch c would be blank (no Coomassie Blue bands); both protein “A” and “B” are absent. Do you think that freeze-fracture followed by SEM would add any useful information about these proteins? Why, or why not? Since neither protein in this system is an integral membrane protein, freeze-fracture would not reveal any additional information about the proteins. It would confirm that neither is integral (both membrane faces “E” and “P” would be smooth (no bumps or dimples)....
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