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arrrgh vesicles

Jane Austen Austen at Bath.UK
Tue Jul 30 10:04:53 EST 1996

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

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