Kinase Assay Question

James Smith jsmith at bioch.ox.ac.uk
Wed Feb 22 09:57:39 EST 1995




I am performing kinase assays on samples of resuspended 18% and 45%
ammonium sulphate precipitates taken from different cell lysates. I am
agonising over two problems:

1.  The Activity is measured in pmol ATP incorperated per minute.  I
normalise this by dividing by the TOTAL protein concentration of the
initial cell lysates (mg/ml) as determined by the BioRad assay
system (OD taken @ 595nm etc).  Ideally, I would prefer to determine the
protein concentrations of the individual 18% and 45% ammonium sulphate
cuts but unfortunately the BioRad system doesn't work with ammonium sulphate.
Does anyone know of any other protein concentration assay system that can
work in the presence of ammonium sulphate?

2.  The buffers I use contain Leupeptin and PMSF as general
protease inhibitors (at 25 and 50 micrograms/ml respectively).  However I
still get degradation of my kinases (PKC's) into their regulatory and
catalytic domains.  Can anyone suggest any other protease inhibitors that
might help prevent this cleavage?

James Smith
Department of Biochemistry
University of Oxford
U.K.








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