Culled from medline:
Biotechniques. 16(2):264-5, 268-9, 1994 Feb.
Purschke WG. Muller PK
Abstract
" We report on a substantial improvement of the widely used fluor
diffusion assay for chloramphenicol acetyltransferase (CAT) activity.
The stable and inexpensive [3H]NaAcetate along with yeast acetyl CoA
synthetase is used to produce [3H]acetyl CoA with high specific
radioactivity and high yield. In a second step, the enzymatically
produced [3H]acetyl CoA is introduced as a substrate for CAT in the
fluor diffusion assay. Due to these modifications, the assay becomes
more sensitive, the range of linearity to produce [3H]acetyl CoA with
high specific radioactivity and high yield. In a second step, the
enzymatically produced [3H]acetyl CoA is introduced as a substrate
for CAT in the fluor diffusion assay. Due to these modifications,
the assay becomes more sensitive, the range of linearity is increased
by two orders of magnitude and the assay becomes less costly."
I personally have done both reactions in the scintillation vial
but I let the labelling reaction incubate for a while before adding the
lysate. Worked well for me ;-) Alot easier than the TLC assays....
Just my $0.02 worth....
Bob Cowherd
<
U. of P.
(215) 662-2071
