western blot stripping protocol

Dr. Kent L. Nastiuk nastiuk at rockvax.rockefeller.edu
Thu Feb 1 19:32:27 EST 1996


In article <4eos1o$obe at mark.ucdavis.edu>,
   szsnoopy at chip.ucdavis.edu (Anthony Martinez) wrote:

>I am having problems stripping off my primary antibody off of my 
>western blots.  I am using nitrocellulose and following NEN's stripping 
>protocol that calls for a 30 minute incubation in 62.5 mM Tris-HCL pH 
>6.8, 2% SDS, and 100 mM 2-mercaptoethanol at 50 degrees centigrade.  My 
>results show that only the secondary antibody is being washed off and 
my 
>primary is still there.  Any suggestions, comments or is there a better 
>protocol to remove the primary antibody off nitrocellulose?
>
>Thanks in advance.
>
>Tony Martinez

you don't mention how long you incubate.

i use the same buffer, but incubate at 70 degrees for 30 min, or 60 
degrees for one hour and it usu works well, *provided* the blot has not 
become completely dry during the first detection.  once it is dry, it 
can be problematic to remove the antibodies

another buffer that others in the lab have used, though i haven't, is 
the same tris/b-me, but substituting 8M urea for the SDS

good luck



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