GST assay

D P Dixon D.P.Dixon at durham.ac.uk
Fri Mar 10 04:52:02 EST 2000


In article <4.1.20000309105223.0099fb00 at pop.mail.nmu.edu>,
John Rebers <jrebers at nmu.edu> wrote:
>Does anyone know of a source other than Pharmacia for GST enzyme assay
>kits? They sell a "detection module" that combines reagents for Westerns
>and a CDNB-based enzyme assay. I have some references for the enzyme assay,
>including Habig et al (1974) JBC 249:7130, but was getting variable results
>and was considering trying a kit if a relatively inexpensive one was available.
>

John,

There really is no need to buy a kit for a CDNB-based GST assay.  Our
group has worked with a variety of GSTs and the CDNB assay has proved  
cheap and straightforward.  I can't remember Habig's method, but we do the
following 1 ml assays:
900 ul 0.1M potassium phosphate buffer pH 6.5
 25 ul 40mM 1-chloro-2,4-dinitrobenzene in ethanol (store in dark at 4 C)
 25 ul enzyme sample
 50 ul 100mM glutathione in water (pH to ~ 7 with NaOH, store at -20 C in
aliquots to minimise oxidation, keep on ice and do not re-freeze).

IMMEDIATELY after adding GSH, measure change in absorbance at 340nm over
say 30s, correcting for the significant rate in the absence of enzyme.  

This should yield highly consistent results - what problems exactly are
you encountering?

Hope this helps,

David Dixon
University of Durham, Department of Biological Sciences
d.p.dixon at durham.ac.uk





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