Stripping nitrocellulose membranes containing phosphoprotein

Fred dynamin at hotmail.com
Fri May 19 21:58:11 EST 2000


We have used the Suck and Krupinska method for a couple of years.  I
have posted it here repeatedly.  Works great.  Sometimes you need to
increase the strip time to 15 min.  Occasionally the first antibody does
not get 100% stripped, but not often.  There will always be a small drop
in signal from the first to second use of the blot, but signal loss
after that is smaller by far than with the SDS method.  We usually use
it for phosphoproteins too (see Wang, X. et al  (1999)  EMBO J. 18,
4549-4559.) and have reprobed up to 10 times (usually only 6 times).

Suck, R.W. & Krupinska, K. (1996) Repeated probing of western blots
obtained from coomassie brilliant blue-stained or unstained
polyacrylamide gels. BioTechniques 21, 418-422. Abstract: No abstract
available.  Wet the dry membrane in water, incubate 5 min in 0.2 M NaOH
at room temperature.  Rinse blot and proceed from blocking step.


Phil
Sydney

Gary wrote:
> 
> I'm trying to strip a nitrocellulose membrane [10 mM b-mercaptoethanol, 0.1%
> SDS, 20 mM Tris, pH 6.5] to restain it with another antibody .  The signals
> I 'm looking for are phosphoproteins. I appear to be losing more signal than
> I would expect.  Is there a chance that I'm hydrolyzing the phosphate group
> during stripping?  How do I avoid this?  Thanks.
> 
> Gary




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