western blot

Heinz-Juergen Schaefers h-j.schaefers at popHost.com
Mon Mar 10 17:07:29 EST 1997

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.
> David Yu

Hi David!

1. block the membrane prior incubation with first antibody long enough 
with TBS/0.1% Tween20, at least 2 hours at room temp or over night at 4 
degrees centigrade.
2. try different secondary antibodies, since some may cause unspecific 
bands or high background
3. wash membrane between each step 2 x 5 mins with TBS/Tween and 1 x 5 
mins with TBS, after incubation with the second antibody wash 2 X 10 mins 
with TBS/tween and 1 x 10 min with TBS.

hope this helps....

Heinz-Juergen Schaefers
University of Erlangen-Nuernberg
Dept. of Medicine IV
Nephrology Research Lab
h-j.schaefers at popHost.com

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