Noam Harel wrote:
>> I am performing immunoprecipitations on unlabeled nuclear
>extracts using non-covalently linked Protein A-antibody combinations.
>The problem is, when I run the precipitates on SDS-PAGE, then do a
>Western blot, the secondary antibody on the W. blot causes the heavy
> chains of the Abs used in the ip to create a
>huge smear that covers up many of the potential bands I'd like to see.
>Is there any way besides covalently linking the Ab to the beads,
>or besides eluting with competitor peptides, to keep the Ab from
>coming off the Prot. A beads when boiling in SDS-PAGE buffer?
>I heard something about elution using glycine, but can't find a protocol
>Thanks for anyhelp,
Try biotinylating the primary antibody used in the western blot and then
use streptavidin (coupled to HRP or AP or whatever) as the secondary
reagent. Then the presence of the IP antibody in the gel is irrelevant. I
have used a kit for biotinylating proteins from Pierce (and also purchased
the streptavidin conjugate from them), but I think lots of companies sell
University of Missouri-Kansas City
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