stayve-and-irayne at worldnet.att.net
Thu Nov 18 01:00:08 EST 1999
Stephen Snowdy <stephen_snowdy at med.unc.edu> wrote in message
news: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...
Generally speaking you don't know until you try it! Start gentle though--no
DOC or SDS. I've had luck with 1% NP40 or Triton X100 in 150 MM NaCl, 50 mM
Tris pH 8, 5 mM EDTA and the usual suspects for protease inhibitors.
Depending on the identity of A,B, and C you may want to try a handful of
detergents and concentrations as well. For starters you might consider
washing the pcpts a few times with the detergent solution above, take an
aliquot for the gel, then wash a few times in the same detergent solution
but increase the NaCl 100mM or so, take a sample, etc.
> Are magnetic beads more efficient than Protein A Sepharose beads?
No experience with magnetic beads and ip's, sorry. I would suggest
comparing protein A agarose or pansorbin with protein G agarose or omnisorb
SEPARATELY rather than mixed. They have different profiles for background
bands that co-pcpt which reminds me...be sure to do a precipitation with the
lysate and beads--no primary antibody. You'd be surprised what can come
down with beads alone!
Johns Hopkins University
stebby at jhmi.edu
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