in vivo phosphorylation
kurbanic at uoguelph.ca
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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