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
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
Look up Methods in Enzymology voolumes 200 and 201 for a variety of protein
Also, please say hi to Ulrike Dohrmann at Univ. Heidelberg....
Toronto Signalling Network: an informal monthly gathering to discuss the latest and greatest in signal transduction.
Information: mailto:jwoodget at oci.utoronto.ca or mailto:m.moran at utoronto.ca
Web site at: http://kinase.oci.utoronto.ca/TSN.html
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