Immunoprecip gurus?

Martin Offterdinger martin.offterdinger at akh-wien.ac.at
Thu Nov 18 05:55:18 EST 1999


Stephen Snowdy <stephen_snowdy at med.unc.edu> schrieb in im Newsbeitrag:
38330FD3.D57A0B38 at med.unc.edu...
> Ok, let's say protein A binds to protein B which binds to protein C.  If
> I pull down with an antibody to protein C (from cell lysates), should I
> expect to be able to pull down protein A with it and probe for protein A
> on a Western, or is it too much to hope for that everthing will stay
> together during the IP?  I'm sure it depends on dissociation contants,
> but generally speaking...
>
> Are magnetic beads more effiecient than Protein A Sepharose beads?
> --
Hi stephen!
Although I m not sure, if you can consider me as an IP-guru, I have quite
some experience with IP
Generally it is possible to do the sort of experiment you have described.
(i.e. Coimmmunoprecipitation). It depends on quite a lot of parameters. You
should carefully choose your detergent (if any) which I am assuming you use
to solubilize your cells/tissue. Some detergents are more aggressive than
others in disrupting protein-protein interactions. Good choices may be:
Digitonin, Brij-series, n-Octylglucoside, CHAPS,...
Rather avoid ionic detergents (DOC, SDS, sarcosinate). Your success also
largely depends on the binding strenght between your proteins A, B and C.
Summarizing this kind of experiments  is rather trial and error as you can
not really predict which detergent may be suited.
Martin






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