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.
>> Simon Penson
> Plant Biology Dept,
> UC Berkeley.
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.