Immunoprecipitation/deglycosylation/western blot?

Kevin Morano kmorano at umich.edu
Tue May 30 21:02:25 EST 2000


In article
<Pine.A41.4.10.10003180226570.134090-100000 at acs6.acs.ucalgary.ca>, Neal
Robert Melvin <nrmelvin at ucalgary.ca> wrote:

> I'm tryin to deglycosylate a membrane
> protein, which I then plan to examine on a western blot to determine what
> proportion of its molecular weight is due to N-linked oligos.....


Dear Neal,

   I did this with a single-pass membrane protein, and what I did was to
IP as usual from a denaturing extract, wash a few times in Triton buffer,
then add the deglycosylation buffer to the beads-antibody-antigen complex,
followed by the enzyme, in this case EndoH.  I think I treated as usual,
then added more denaturing buffer, boiled, diluted out, and re-IP'd.  You
don't have to do this last, probably, just spin out and r/s in sample
buffer.  Bottom line - you should be able to deglycosylate on the beads.

Kevin






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