Kinase Assay Question

James Smith jsmith at bioch.ox.ac.uk
Tue Feb 21 09:30:17 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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