Immunoprecipitation and subsequent phosphotyrosine western

John Hines hines at pharm.med.upenn.edu
Thu Feb 10 17:34:44 EST 2000


In article
<Pine.A41.4.10.10002052252350.91100-100000 at acs6.acs.ucalgary.ca>, Neal
Robert Melvin <nrmelvin at ucalgary.ca> wrote:

> Is it necessary to have orthovanadate present in my solutions after I've
> transferred my proteins to my membrane?? For example, should it be present
> in my blocking solution, and in with my primary antibody?
> 

Neal,

    I don't think you will find it necessary to include phosphatase
inhibitors in your blocking solution or any subsequent steps.  You should
get plenty of immunoreactivity without them.  If you find that
orthovanadate is insufficient to preserve immunoreactivity, I would
recommend adding supplementary phosphatase inhibitors to your cell lysis
buffer before anywhere else.  You might try phenylarsine oxide (at 1 um --
make a higher concentration stock up in DMSO), or sodium molybdate (at 1
mM in the assay) or even NaF and Na pyrophosphate.


   If you are using alkaline phosphatase as your developing step in the
blotting process, that would be another reason to stay away from
phosphatase inhibitors in your wash solutions.


John




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