Acetyl CoA for CAT

[Robert Cowherd]

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
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