easy enzyme assay

Tom Deits 21664mgr at msu.edu
Mon Jan 10 10:48:00 EST 1994


In Article <2gk8j5$fp4 at dartvax.dartmouth.edu> "bob.gross at dartmouth.edu (Bob Gross)" says:
> Hi folks,
> 
> I am involved in teaching a large introductory course in Cell Biology,
> which includes a lab. One of the labs is a molecular sieve column with
> three standard proteins and one unknown. Fractions are collected
> manually and protein peaks and assayed by a Bradford assay. With the
> equipment we have, it is easy to resolve three peaks, but adding a
> fourth becomes very problematic. 
> 
> Does anyone know of an inexpensive enzyme that has a very simple
> colorimetric enzyme assay that could be used to detect trace amounts of
> an unknown in the presence of the large amounts of standard proteins
> which are detected by Bradford?
> 
> Thanks in advance for any suggestions.
> 
> Bob Gross
> Dept. of Biology, Dartmouth College
> bob.gross at dartmouth.edu
> 
You might want to consider carbonic anhydrase, using p-nitrophenyl acetate
as substrate.  Make up the PNA stock in dry acetone, and it is quite stable.  
CA has the advantage that it is a commonly used MW standard, so you can expect
it to behave reasonably on your sizing column.
Tom Deits
Dept. of Biochemistry
Michigan State Univeristy
21664mgr at msu.edu



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