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
