IP-Western

Ian A. York iayork at panix.com
Fri Jan 19 10:13:37 EST 2001


In article <20010119150944.5BDD7415D5 at mercury.hgmp.mrc.ac.uk>,
 <huangz at biowww.net> wrote:
>In an immunoprecipitation-western blot I used rabbit polyclonal antibody
>and protein A agarose beads(protein A is covalently bind to beads) to
>pull down antigen complex. In following western blot I used rabbit
>polyclonal antibody as primary antibody and HRP-anti rabbit IgG as
>secondary. The result turns out to be many bands, including IgG bands.

I've never seen a perfectly clean IP-western, but I've done a fair few 
that are pretty good.  If you blot with an antibody from a different 
species than the IPing antibody, and probe with a species-specific 
antibody (not just an antibody raised to one species, which generally 
cross-reacts; you need to use  an antibody that has been actually stripped 
of cross-reacting species), you can get clean results.

If you absolutely have no choice and must use two rabbit antibodies, you 
might try covalently coupling the antibody to beads, instead of using 
protein A; I've never tried this, but it might help, at least with the 
heavy chain cross-reactivity.

Ian 
-- 
    Ian York   (iayork at panix.com)  <http://www.panix.com/~iayork/>
    "-but as he was a York, I am rather inclined to suppose him a
     very respectable Man." -Jane Austen, The History of England






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