[Bioforum] Re: immunoprecipitation problems
Dr Engelbert Buxbaum
(by engelbert_buxbaum from hotmail.com)
Wed Jan 16 11:50:36 EST 2008
Am 05.12.2007, 10:42 Uhr, schrieb Gu, Hongbo <Hongbo_Gu from brown.edu>:
> I am wondering is there something wrong with sample preparation, I added
> the lysis buffer to cells, sonicate them briefly, incubate on ice for
> 1h, centrifuge and pre-clear it by preimmune IgG and protein G bead,
> after all above steps, the receptor still present by western, but
> cannnot be pulled down by IP.
When you detect proteins on a Western, they are denatured and a
successfull antibody is usually directed against a peptide sequence.
To immunoprecipitate a protein, the antibody needs to recognise the native
protein, and may be directed against a structural epitope (aa which are
close to each other in space, but far appart in primary structure).
Therefore you may need 2 antibodies, one to precipitate and one to detect
on westerns. The suppliers docs should state for which purpose your Ab are
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