Western blot vs. immunoprecipitation??

Ian A. York iayork at panix.com
Wed Nov 15 08:49:26 EST 2000

In article <Pine.A41.4.10.10011150108520.22296-100000 at acs4.acs.ucalgary.ca>,
Neal Robert Melvin  <nrmelvin at ucalgary.ca> wrote:
>What would the likely reason be behind the finding that often a particular
>antibody shows a lot of non-specific bands on a western, but only the
>ones known to be specific after an IP?? Do antibodies have higher

This is not uncommon.  I don't think it's a matter of solution vs.
attached; I think it's more likely because proteins are denatured during
the SDS-PAGE and transfer to substrate.  Two results--first, the proteins
present different appearances to the antibody, so that a protein that
doesn't specifically cross-react in solution may now cross-react; second
(and probably more important) because the denatured proteins present more
hydrophobic faces, the chances of *non-specific* interactions are higher.

A useful point is that if the new interactions are non-specific, you may
be able to overcome the problem by adjusting your blocking buffer and
binding conditions.

A third possibility, also important, is that in the western you're seeing
interactions that are present in the IP, but are invisible, because in the
latter the proteins are not metabolically labelled--components of culture
medium such as albumin, for example.  If that's the case (if a major
contaminant band runs at arounf 65 kDa), be sure to wash your cells before
lysing them, to get rid of the serum in medium; it makes a big difference
in western quality.

    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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