Anti-serine phosphatase

Jim Woodgett 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 
61-8100.  

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 
ser-specific phosphatases).  

Jim Woodgett


(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. 
>  
> Thanks. 
>  
> Ian 


Associate Professor: University of Toronto
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