reprobe western blots

Robert Bacallao nephron at merle.acns.nwu.edu
Thu Jan 12 12:31:28 EST 1995


In article <3eg2t9$8hl at neuro.usc.edu>, william at neuro.usc.edu (Fiberman) wrote:

> Hello, Netters,
> 
> Can a western blot filter be "reprobed" like a Southern blot filter?  If
> so, what is the best way to strip the original antibody?  I am using a
> secondary antibody conjugated to alkaline phosphatase, and visualizing
> with addition of color substrates NBT and BCIP.  I would appreciate your
> response.
> 
> -fm

This is the first time I have replied to a net question so please excuse
me if I make a faux paux.  Yes, you can reprobe a wester blot.  Incubate
the blot (under constant agitation) at 65 degrees C for 1 hour in 2% SDS,
62.5 mM Tris-HCL, pH 6.8; 100 mM beta-mercaptoethanol.  We do this in a
hood for obvious reasons.  This method was first described by Kaufmann
(1987) Analytical Biochem. 161., 89-95.  

Good luck


nephron



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