p53 western blot

Jes Kuruvilla jkuruvil at lamar.colostate.edu
Wed Jun 20 12:10:02 EST 2001

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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