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Query: EC:2.3.1.28 (
chloramphenicol acetyltransferase
)
5,100
document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)
We describe a simple, rapid yet extremely sensitive assay for
chloramphenicol acetyltransferase
(
CAT
) activity in extracts from transfected eukaryotic cells. Using our modified reaction conditions and the mixed-phase assay, less than 0.000010 unit of
CAT
activity in transfected cells can be reliably detected. The mixed-phase assay is based on the inability of the polar [3H]-acetyl-Coenzyme A (
CoA
) substrate to partition out of a urea containing aqueous phase into the nonpolar scintillation fluor, while the [3H]chloramphenicol reaction products partition into the toluene scintillation fluor and are quantitated by scintillation counting. The increased sensitivity of this assay is due to the optimization of the acetyl-CoA concentration, to a urea-containing aqueous phase which lowers the assay background, and to the use of extract blanks. The mixed-phase assay is simpler, is quantitative, uses less costly substrates, and is far more sensitive than the most widely used
CAT
assays, which require solvent extraction followed by thin-layer chromatography to separate the unreacted substrate from product.
...
PMID:A highly sensitive, mixed-phase assay for chloramphenicol acetyltransferase activity in transfected cells. 275 94
