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?
How about in-vitro translation using rabbit reticulocyte lysate?
I've recently purchased lambda phosphatase and am wondering how to dephosphorylate cell lysates. Do you mind sharing your protocol?