Stripping Western blots

Ann Sutherland via methods%40net.bio.net (by as9n from virginia.edu)
Fri Oct 26 12:31:59 EST 2007


Hi,
You're right - better to describe the already tested methods.  The 
methods we have tried are:

1.  1% SDS in PBS overnight at 37degreesC.  This has variable results 
depending on the antibody, but doesn't result in much loss of protein.

2.  2% SDS, 100mM beta-mercaptoethanol, 62.5mM Tris pH 7.4, for 30 
minutes either at 50 degreesC or at room temperature.  This works for 
blots of cultured cell lysates, but results in high loss of protein 
so it is too harsh for blots of embryo protein.

We're looking at the degree of phophorylation of several proteins, so 
we need to have good removal of antibody, but we have very little 
protein, so we need to minimize protein loss.
Thanks,
Ann



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