I'm going to isolate vesicles from bacterial supernatant and run western to see if my protein was incorporated into them. After ultracentrifugation I obtained pellet which supposedly contain vesicles. Should I resuspend it in PBS with SDS loading buffer, boil and run the gel?
Can I quantify protein content prior boiling by nanodrop? In this case how do I break vesicles to release proteins?
Can you use RIPA buffer to resuspend the pellet (just like the preparation of cell lysates for WB), this way the membrane of the vesicles is disrupted? And do the BCA assay to quantify protein concentration, with RIPA buffer as the background. (FYI, RIPA buffer does not give background in BCA assay in my hand).
1) First aim should be to detect your protein in the pellet. No need for PBS, I would directly add SDS loading buffer + DTT to the pellet, heat to 95 degrees Celsius for 5 minutes, and load on gel twice, once for coomassie staining or silver staining, the other for western blotting. This will give you a starting point.
2) If you cannot detect your protein, then you have to rethink everything.
3) If you can detect your protein, then you have an estimate from the gel staining how many other proteins are present. Then you can try repeating the procedure, but this time you sonicate the pellet with PBS first, measure protein concentration, load a specific amount on the gel and detect with western.
4) However, to determine if your protein is present in vesicles (from a bacterial supernatant) or in large molecular weight protein aggregates is harder, you would have to do a protease protection experiment. Bacteria usually don't produce vesicles in the supernatant, bacteria don't have a secretory pathway with individual membrane compartments either, so this is either a very innovative line of research, or perhaps there is confusion about the term vesicle.
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