Hi,
I have been trying to incorporate a solubilized transmembrane toxin protein into eukaryotic cell membrane. Upon incubation with vesicles the protein form pores and ruptures the vesicles as reported earlier and also seen during in house trials. Is it possible to obtain protein-incorporated-membrane-fractions (detectable in SDS-PAGE) if I incubate the purified protein with yeast spheroplast (harvested using lyticase and sorbitol) ?
You should be able to see how much of the protein is incorporated into the spheroplast membranes by pelleting them in an ultracentrifuge and running them on SDS-PAGE.
Yes Adam,
That's what I'm trying to get. But I was worried about the yield of incorporated protein. Do you think that yield would be convenient in this procedure? Is it anyway possible that the proteases of yeast cells will disrupt the membrane-incorporated proteins?
The fact that the protein causes rupture of the vesicles works against you as far as proteolysis is concerned. If you include a protease inhibitor cocktail, you may be able to minimize proteolysis of the attached protein. If you purify plasma membranes from the cells by sucrose gradient centrifugation, you should be able to reduce proteolysis and improve the sensitivity of the method. If Coomassie staining is insufficient to detect the bound protein, you could use Western blotting to increase the sensitivity.
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