Antibodies to phosphorylated amino acids

Dennis Goode GOODE at ZOOL.UMD.EDU
Wed Jan 25 17:00:24 EST 1995


Simon Penson with swanson at nature.berkeley.edu (Sarah Swanson)
on   Mon, 23 Jan 1995 09:01:23 wrote
> I'm looking at the possibility of using antibodies to phosphorylated amino
> acids as an alternative to radiolabelling in studies of protein
> phosphorylation. Has anyone got any tips/comments about this, especially
> regarding the specificity of the antibodies.
> 
> Thanks,
> 
> Simon Penson
> Plant Biology Dept,
> UC Berkeley.
> 
 Simon:
 Antiphosphotyrosine is widely and successfully used, especially for 
immunoprecipitation and Western Blotting.
 My student Bob Wheelock has tested a number of different 
commercially-available antibodies and found that monoclonal 4G10 from 
Upstate Biotechnology was the most efficient at 
immunoprecipitating and blotting phosphoproteins from mouse and human 
cell lines. Proteins can be eluted from precipitates easily and 
gently with phenyl phosphate. WRT specificity, we do occasionally 
find a band or 2 on gels that antiPT binds to even in the presence of 
phenyl phosphate, which is one control for specificity.

I understand that antiphosphoserine and threonine are starting to be 
used, but so far with less success. I'd like to hear if others are 
finding them useful.

-Dennis  




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