in vivo phosphorylation

Kevin Urbanic kurbanic at
Mon Apr 24 11:08:29 EST 2000

I am interested in testing for in vivo phosphorylation activity of a 
bacterial protein I have cloned into pET-28a. I plan to use P32-labeled 
H3PO4 (purchased in 0.02N HCl, ICN) for these exeriments.  

Does anybody have a proper procedure for experiments similar to this.  
Will the radiolabeled H3PO4 diffuse through the bacterial cell (E. coli 
DH5)?  Is the Histidine-phosphorylated protein (a histidine kinase) stable 
enough to withstand alkaline cell lysis followed by suspension in 
SDS-PAGE loading buffer?

Your suggestions and general help would be greatly appreciated. 


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