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