[Bioforum] Re: immunoprecipitation problems

[Dr Engelbert Buxbaum]

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  
suitable.