Kinase assays of in vitro translated protein, suggestions?

Nick Theodorakis nicholas_theodorakis at urmc.rochester.edu
Wed Jun 6 08:25:40 EST 2001


In <3B1E180E.4C68AEB6 at hgu.mrc.ac.uk>, Graham Dellaire wrote:
>
>Dear All,
>
>I am attempting to use in vitro translated protein for kinase assays using 
immune complexes from HeLa cells containing a ser/thre kinase.  Has 
anyone out there tried this or seen a paper where this was done?  I am 
thinking of
>either using an aliquot of in vitro translated protein directly in my assay or 
first immunoprecipitating the protein with specific antibodies, followed by 
mixing of the kinase and target protein immune complexes in kinase 
buffer
>(i.e. the protein A/G beads and protein complex).

I haven't done ivt/kinase reaction together, but I might suggest to 
immunoppt. the protein first because most ivt reactions have large 
amounts of ATP, as well as an ATP regenerating system, which might 
make it diificult  to perform a kinase labelling in a crude lystae.

Nick


-- 
Nick Theodorakis
nicholas_theodorakis at urmc.rochester.edu

 




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