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specific phosphorylation - (Feb/19/2013 )

I am interested in the target of one specific kinase.
So I was thinking of dephosphorylating cell lystae (which shouldn't be a problem) and than to incubate the dephosphorylated lysate with my kinase and see what is re-phosphorylated.
of course this means that my proteins must be functional after dephophorylation (so no boiling, dtt etc), and ideally, but probably nearly impossible, to supress all kinases except the one I am adding.

Did anybody ever did this? or heard of somebody doing this?


-simone dorfmueller-

How about in-vitro translation using rabbit reticulocyte lysate?


HI SImone,

I've recently purchased lambda phosphatase and am wondering how to dephosphorylate cell lysates. Do you mind sharing your protocol?

-Joe KooL-