western blot

Alix Helme-Guizon ahelme at lovelace.infobiogen.fr
Tue Mar 18 07:05:06 EST 1997



On Mon, 10 Mar 1997, Heinz-Juergen Schaefers wrote:

> David Tak Yan Yu wrote:
> > 
> > I have been trying to do western blot with antibodies against immediate
> > early genes eg c-fos.  There is quite a lot of background.  I would
> > appreciate if advices.
i've had a lot of back ground with westerns for two different reason:
1) the first antibody, from Sigma, was a real sh.....purification doesn't
improve the specificity. Had to buy another one.
2) with a good first antibody, the crucial step for background is the
washing after incubation with yhe secondary antbody. 
i wash at least five times in TBS-tween and milk, and at least 20 min per
washing. don't be affraid of "wasting" an whoole afternoon only for
washing the membrane. you will be payed back!

good luck!
Alix 




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