Phosphorylation Assay Conditions
szmazer at chip.ucdavis.edu
Mon Jul 24 14:20:50 EST 1995
Cornelius Krasel (krasel at wpxx02.toxi.uni-wuerzburg.de) wrote:
: 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.
You are correct. But it might be helpful to actually say that the "hot"
ATP should have "cold" ATP as a carrier. The total concentration should
be 100 mM as you said. (Don't forget Mg2+). It is generally true that in
vitro doesn't always translate into in vivo, but that's where enyzme
kinetics comes in. It might be helpful to vary the substrate
concentration and determine the Km value. If the Km value is close to the
intercellular concentration of the substrate then it might likely be
real. Further analysis will be required. Good Luck.
: /* 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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