S35 Pulse Chase analysis of an endogenously expressed glycoprotein

Vladimir Gainullin via methods%40net.bio.net (by gainullin from gmail.com)
Fri Aug 17 00:55:29 EST 2012


Hello,  I am able to enrich and immunoprecipitate a very large glycoprotein
from human epithelial cells. I suspect that it undergoes  ectodomain
shedding as I can detect it in the media. To analyze trafficking I
attempted to use S35 labeling which repeatedly failed with only background
bands showing up on the radiogram.  Various pre-clearing steps to reduce
background did not help. Is it possible that S35 pulse chase is simply not
suitable to study proteins expressed at very low levels? Does Cycloheximide
chase substitute for S35 pulse chase?

Thank you.


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