jwoodget at oci.utoronto.ca
Fri Mar 7 10:23:37 EST 1997
The best way is the 32P label it (by metabolically labelled cells and immuno-
purifying the protein) and perform a phospho-amino acid determination.
Failing that, Zymed <http://www.zymed.com> have some supposedly more specific
polyclonal antibodies than Sigma (I haven't tried them though). Cat number
Bear in mind that many proteins contain some phospho-serine so it wouldn't be
too surprising to find it. As for a control protein, casein is serine,
threonine and tyrosine phosphorylated (even the partially hydrolysed stuff).
Commercial preps of histone are also phosphorylated to varying extents. A
control would be to treat your protein with a serine/threonine phosphatase
prior to blotting. Phosphatases 1 and 2A are commercially available.
However, they both dephosphorylate both serine and threonine (there are no
(In article <5fmupd$r23 at panix.com>, iayork at panix.com (Ian A. York) writes:
> I need to see if a protein is serine-phosphorylated. I'm going to use
> Sigma's anti-phosphoserine antibody (IP my protein, then blot it and
> probe with the anti-ps), and I'm aware that it's pretty pathetic. Does
> anyone have a suggestion for a nice control protein? Ideally it would be
> moderately abundant, constitutively serine-phosphorylated, easily
> immunoprecipitated with a readily available antibody, and not either 25
> or 50 kDa, because if it is the antibody on the blot will overlay it.
> Also, if anyone has any tips for making the anti-phosphoserine antibody
> more reliable, I'd appreciate hearing about it.
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