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!

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