western blot

Rupert Yip yip at rascal.med.harvard.edu
Mon Mar 10 20:07:44 EST 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.
> >
> > 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....
> 
> bye
>         Heinz
> --
> Heinz-Juergen Schaefers
> University of Erlangen-Nuernberg
> Dept. of Medicine IV
> Nephrology Research Lab
> h-j.schaefers at popHost.com


Is there any reason why TBS is used over PBS?  Is one superior to the
other?



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