p53 western blot

Nora Plesofsky nora at biosci.cbs.umn.edu
Wed Jun 20 12:36:23 EST 2001

Another important variable is the concentration of primary antibody 
that is used. I found that reducing this concentration (while still 
allowing detection of antigen, of course) goes a long way toward 
reducing background without changing washing conditions.


>Hi Cris,
>My protocol uses the DO5 Ab,
>Bind Ab to the blot in Wash Buffer {(10% non fat Milk in
>PBS(ph7.4) with 10% Tween 20)} for 2 hrs or more.
>Wash with wash buffer for 20min each 3X
>Add 10 ml of PBS(1X) for 45 min along with secondary Ab.
>Rinse 3X quickly with 5ml of PBS
>Then wash with PBS 3x for 10 min each
>Allow to air dry and then probe with ECL plus.
>Air dry and scan over a STORM instrument or place over
>appropriate film.
>cris wrote:
>>  Hi, i'm looking for a good protocol for p53 western blot. My problem is a
>>  very high background on nitrocellulose membrane after chemoluminescent
>>  reaction. We use very striking washing conditions, but they are not enought.
>>  Please help me. thanks
>>  <http://www.biowww.net/forum/read.php?f=1&i=2595&t=2595>


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