Immunoprecip gurus?

Ian A. York iayork at
Fri Nov 19 18:02:29 EST 1999

In article <811qdj$fbe$1 at>,
Dima Klenchin <klenchin at> wrote:
>: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
>My experience with IP is that if one tries really hard, it is possible
>to co-IP anything with anything. Particularly when a criterion for 
>co-IP is a sensitive western. And that is the major problem with
>this overused undervalidated technique.

This is absolutely true, but there's nothing intrinsically wrong with the
technique.  It's just that you ABSOLUTELY MUST do the appropriate 
controls.  A student working for me is doing this right now, and I
insisted on seeing multiple different antibodies that do and do not bind
the protein; the same antibodies in cells that don't express the protein;
and much more.  We started out seeing four proteins that apparently
specifically co-precipitated, and only by doing the full series of
controls were we able to determine that three of the four are

Other people have offered good sugegstions, like playing with different
detergents (in order of increasing stringency, I use digitonin, CHAPS,
NP40, NP40/DOC, NP40/DOC/SDS).  You can also play with salt concentration
(low salt is often less stringent, but not always; it depends on the
nature of the interaction).  But don't be TOO gentle, or you'll almost
certainly get non-specific interactions.

    Ian York   (iayork at  <>
    "-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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