Kinase Assay Question

Giovanni Maga maga at vetbio.unizh.ch
Thu Feb 23 11:05:15 EST 1995


In article <Pine.SOL.3.91.950222145600.4000B-100000 at topaz.bioch>,
jsmith at bioch.ox.ac.uk (James Smith) 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?
> 
> 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.

You can use also Chemostatin, Pepstatin and Aprotinin, they are sold by
almost all big companies (Sigma, Boehringer, etc.).
About the problems of protein det.: I got that you are using the
resuspended pellets. So, after AS precipt. you should resuspend your pellet
in low AS buffer, isn't it? Then why don't you dialyze your samples (or
part of them) in order to get rid of all AS (given that the final conc. of
AS after pellet resusp. is still too high)? If the volumes are too small to
be dialyzed, you can also exchange your buffer by some desalting column.
Moreover I do not think that it is a good idea to perform enzymatic assays
in the presence of high salt (unless you think that AS does not interfere
at all with your activity).
Good luck. G.Maga.
maga at vetbio.unizh.ch



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