tearing my hair out over IPs

[ijiwaru at wheel.dcn.davis.ca.us]

In article <316C5BAF.56CC at genetics.utah.edu>, Jen Logan
<jlogan at genetics.utah.edu> wrote:

> Hello,
> 
> I am trying to use co-immunoprecipitation to confirm an interaction 
> between my favorite mammalian protein and a putative binding partner.  
[stuff deleted]
> Approaches/reagents that have failed:
> 1) Both protein A sepharose and protein G agarose give the same 
> results,  even after blocking in 1% BSA.
> 2) Preclearing the lysate with protein A/G for 1 hour at 4° C doesn't 
> help.
> 3) Lysis/wash buffers with higher salt (up to 250 mM KCl) and 
> detergent (0.1% NP-40, 0.1% Triton X-100, 0.1% SDS) don't make any 
> difference,  but neither do they disrupt immunoprecipitation. 
> 4) Even "high stringency" washes in RIPA buffer (1% deoxycholic acid, 
> 0.1% SDS, 0.1% Triton X-100) do not disrupt precipitation of the 
> candidate binding partner.
> 5) My standard lysis/wash buffer is PBS + 0.1% NP-40,  with the above 
> goodies thrown in.
> 
[more stuff deleted]
One thing we used to do to try and get rid of everything but our
'specific' band of interest in IPs was to run a high salt wash (1M MgCl2)
followed by a low salt (10mM something).  It was good at getting rid of
actin, if my memory serves me correct.  Since you are looking for co-IP,
YMMV.

One thing you could also consider is a "Far Western" approach whereby you
in vitro translate one of your proteins in the presence of label and use
that to probe a blot with your cell extract.

Good luck,

Lyle Najita
Plant Pathology
University of California - Davis