Western blot/antigen

[Gopal Thinakaran]

Hi eveyone,

	I have a couple of questions regarding ways to treat proteins bound to 
nitrocellulose/nylon membnranes:

1)  I have used strips of NC/nylon membrane containing .5 to 1 mg of GST 
fusion protein to affinity purify antibodies. I notice that some of the 
fusion protein comes off the membrane during the affinity purification 
process.  Is there a simple way of treating the membrane after protein 
transfer to "fix" the protein.  (I am not sure how reliable drying the 
nylon membrane is).

2)  One of the anti-peptide polyclonal antibodies that I am currently 
characterizing works well for immunocytochemistry (4% para fixation for 
upto overnight) on brain sections.  The staining appears to be real 
since it is completely blocked by prior incubation of the antibody with 
the peptide antigen.  However, I am having difficulties detecting the 
protein on blots.  Is is reasonalble to attempt denaturing the protein 
(or treat the blot) by incubation with urea or guanidine to "expose" the 
antigenic site?

Thanks,

Gopal.

Gopal Thinakaran, Ph.D
The Johns Hopkins University School of Medicine
Baltimore.