Co-Immunoprecipitation of nuclear receptor

Hi everyone,

I'm presently having problem to do my co-immunoprecipitation. I
immunoprecipitate a nuclear receptor and I'm trying to look at the
proteins that are associated to that receptor. The problem is that,
once actif, my receptor become highly link to DNA, so it become
insoluble and the condition that affectes its DNA-protein interaction
also distroyes the protein-protein interaction with my protein of
interest. I was wondering if I could crosslink my proteins before the
immunoprecipitation (to have the active form of my receptor) and look
at it after by SDS-Page ? Does the condition of an SDS-page is harsh
enough to break all the protein-protein interaction of crosslink
proteins so that I can look by western blot at my protein of
interest ?

Thank in advance,

Nath

If you use formaldehyde as crosslinker, you can break the interaction
by heating. You can compare the sample with and without heating.
Without heating your complex will be intact. One thing keep in mind
is once you do crosslinking, it will be really hard to extract the
protein that you are interested in. You'd better do time course to
optimize the condition for crosslinking.

Zhicheng

At 05:24 PM 3/4/2007, Nath wrote: