Kinase Assay Question

Wed Feb 22 17:43:53 EST 1995

James Smith
Department of Biochemistry
University of Oxford writes:

>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?

How about the old fasioned absorbance at 260/280?  Watch out for detergents that 
contain aromatic rings eg NP40.  Amino acid analysis would give accurate figures.

>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?

I like E64 (10uM) as a stable irreversible inhibitor of cysteine proteases  and AEBSF 
as a fairly stable (months) irreversible inhibitor of serine proteases.  Note that E64 
only reacts with active proteases so if you later add DDT or 2ME in the absence of E64 
you can activate cysteine proteases that have not reacted with E64.

All the best, 
Martin Slade,
School of Biological Sciences,
Macquarie University,
NSW 2109,
FAX  (61 2) 850 8174
Phone(61 2) 850 8210

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