Kinase Assay Question

Curt Ashendel ashendel at aclcb.purdue.edu
Wed Feb 22 15:01:05 EST 1995


On Tue, 21 Feb 1995 14:30:17 +0000, 
James Smith  <jsmith at bioch.ox.ac.uk> wrote:

>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?

I think the Lowrey assay (also the BCA version) should tolerate this, but I 
can't remember with certainty. If your samples are sufficiently 
concentrated you may be able to dilute the sample with water or weak buffer 
to reduce the AS conc until it can be tolerated by the Bradford reagent. 
Be certain use appropriate standards and blanks if you do this.

>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?

Aprotinin.

Also, making more dilute lysates helps increase stability to proteases. In 
my hands PKC degrades in lysates even at -20 degrees, so the assay must be 
done immediately and native samples must be kept cold at ALL times.

Sorry, but PKC is just comparatively sensitive to proteases.  


Good luck.

Curt Ashendel
Purdue University
West Lafayette, IN
ashendel at aclcb.purdue.edu



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