Phosphorylation protocol

Jim Woodgett jwoodget at oci.utoronto.ca
Thu Jul 25 18:26:31 EST 1996


In article <4t5io4$8fq at sun0.urz.uni-heidelberg.de>, Christoph H. Winter <
chw at sirius.mgen.uni-heidelberg.de> writes:
 
> I?m looking for a protocol for the non-specific phosphorylation of 
> a recombinant protein with the SIGMA protein kinase catalytic 
> subunit (#P2645, does not need cAMP activation) or a comparable kinase. 
> I would be very grateful for some advice and your experience. 

Easy, just provide a buffer around pH 7.2 (+/- 0.5), some magnesium ions (2-5 
mM) and 32P-ATP labelled at the gamma phosphate position (final conc. 50-100 
uM, spec. act. about 100 cpm/pmol or so).  Incubate with your protein 
substrate for 5-30 min. at 30-37 deg C and run on an SDS-PAGE gel.  Dry and 
expose.  Conditions are deliberately vague as all thats really necessary is 
MgATP.

Of course, your protein may not be labelled very well if it doesn't contain a 
decent consensus for cyclic AMP-dependent protein kinase (Arg/Lys-X-Ser/Thr is 
one target).

Look up Methods in Enzymology voolumes 200 and 201 for a variety of protein 
phosphorylation assays/conditions.

Also, please say hi to Ulrike Dohrmann at Univ. Heidelberg....

Jim Woodgett


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