I have another question concerning sec vesic le isolation. I am
currently using the Goud et al. vesicle floatation protocol but I am
having troubles with proteolysis after lysing spheroplasts. I have
been generating spheroplasts with zymolyase in sorbitol buffer and
pelleting the spheroplasts through a 1.7 M sorbitol cushion. I then
lyse the spheroplasts in 0.8 M sorbitol / TEA buffer in the presence
of PMSF, pepstatin A, leupeptin & aprotinin. I also tried adding
chymostatin, but to no avail. Certain membrane proteins are still
getting clipped. I have traced the source of protease to the
zymolyase so whatever is in there seems to be sticking to the pelleted
spheroplasts. Does anyone out there have any suggestions for the
correct inhibitor to add?
Neil Rowley, MIT dpt of Biology
