Methods Digest, Vol 22, Issue 4

Qin Su via methods%40net.bio.net (by qsu from sanger.ac.uk)
Mon Mar 5 13:11:27 EST 2007



Message: 4
Date: 4 Mar 2007 14:24:06 -0800
From: "Nath" <picardnath from hotmail.com>
Subject: Co-Immunoprecipitation of nuclear receptor
To: methods from net.bio.net
Message-ID: <1173047046.477929.165090 from j27g2000cwj.googlegroups.com>
Content-Type: text/plain; charset="iso-8859-1"

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


Just a thought, never tried though. How about slight sonication to shear
the DNA if you are sure the protein is attached to DNA rather the
insoluble cytoskeleton? And also, I seem to have heard of de-crosslink
the proteins, but can't remember where, if you can find out, it should
solve your problem. The crosslink of protein should be covalent bond(not
disulfite bond), is the treatment for SDS-PAGE capable to break it?
Would like to know how you work it out.





More information about the Methods mailing list