Phosphatase protocol for Westerns

Bernard Murray, PhD spam at 127.0.0.1
Fri Jun 23 22:32:54 EST 2000


In article <3951DB6A.5D55CEC7 at hgu.mrc.ac.uk>, G.Dellaire at hgu.mrc.ac.uk
("G.Dellaire") wrote:

> I am
> having a tough time finding any protocols for phosphatasing proteins for
> Western analysis, although there are many protocols available for
> inhibiting phosphatases.  Does anyone have a good protocol?

Sorry no, but I understand potato starch phosphatase is suitable. 

> Ideally it would be nice to selectively phosphatase tyrosines versus
> serine/threonines, in addition to blanket dephosphorylation.

There's an alkali treatment which selectively removes phosphate from
S/T and spares Y.  I don't have it to hand but I seem to remember
cooking it in 5 M KOH (you have to use PVDF).  Since most of the
classic phospho-X antibodies are *selective* rather than *specific*
this is a good test when using these reagents.
 
> Also if I want to preserve the phosphorylation status of the protein
> during extraction, should I be adding phosphatase inhibitors to the
> cells before harvest as well as during the extraction?

No, just work quickly.  If you add the inhibitors to the cells you may
alter the phosphorylation status.

   Bernard

-- 
Bernard P. Murray, PhD
bpmurray at cgl . ucsf . edu
Department of Cellular & Molecular Pharmacology, UCSF






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