Phosphorylation Assay Conditions

Cornelius Krasel krasel at wpxx02.toxi.uni-wuerzburg.de
Sat Jul 22 05:08:07 EST 1995


bmbrl at biovax.leeds.ac.uk wrote:
: I'm in the process of setting up an assay system to see if a membrane protein
: I've purified is a substrate for cAMP-dependent protein kinase (PKA).
: Firstly, what concentrations of ATP should I be using?  If I add [32P]ATP to
: the reaction mixture the final concentration will be ~0.1uM.  Would this be
: enough to indicate preferred substrates for phosphorylation?  

For PKA phosphorylation of a soluble protein I used a final concentration
of 0.1 mM ATP with an activity of roughly 1E6 cpm. I think if you add
only radioactive ATP the ATP will become quickly the limiting factor.

Be aware of the fact that a phosphorylation in vitro does not necessarily
mean anything in vivo.

--Cornelius.

--
/* Cornelius Krasel, U Wuerzburg, Dept. of Pharmacology, Versbacher Str. 9 */
/* D-97078 Wuerzburg, Germany        email: phak004 at rzbox.uni-wuerzburg.de */
/* "Science is the game we play with God to find out what His rules are."  */



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