Phosphorylation of Proteins - Detection?

Pierre Hubert hubert at cigale.u-strasbg.fr
Fri Jul 15 03:48:50 EST 1994


>In article <2vitui$q7q at dingo.cc.uq.oz.au>, forrest at biosci.uq.oz.au
(acer) wrote:
>> G'day all,
>>         Does anyone know of a NON-radioactive procedure for testing
whether a 
>> protein is phosphorylated or not? 

>In article <Stephen_Lasky-1307941642020001 at tonto-slip6.cis.brown.edu>
Stephen R. >Lasky, Stephen_Lasky at brown.edu answers:
>You might try western blotting with antibodies against phosphotyrosine
>(which I know are available) or phosphoserine or threonine (which I think
>are now available).  Then detect with ECL.  We have done this with anti
>P-Y antibodies and the technique is VERY sensitive.  To be even more
>specific you could immunoprecipitate your protein with specific
antibodies
>before you run it on the gel for a western.  Hope that helps.
>
	Yes, Western blot after immunoprecipitation (or not) would be the
non-radioactive method of choice if you have little material to start
with. Unfortunately, commercial anti-phosphotyrosine antibodies often
display quite different specificity. And antiphosphoserine and -thr are
sold by Sigma, but I have not seen lately any publication mentionning
their use ??
	One *LESS*-radioactive method would be to use 33P-orthophosphate instead
of 32P to perform whole cell-labelling. I have been dreaming of doing
that, 33P is much less dangerous to work with than 32P, but is awfully
more expensive... Has anybody some experience with 33P labelling of
proteins?
	Hope this is of some help.

	Pierre HUBERT
	INSERM U 338
	Strasbourg	France
	hubert at cigale.u-strasbg.fr



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