Sensitive radiometric assay for chloramphenicol acetyltransferase using automated HPLC.
Chloramphenicol acetyltransferase (CAT) is widely used as a reporter element in studies of eukaryotic gene expression. We report a new technique for measurement of CAT activity that incorporates several advantages over the existing procedures from which it is derived. The combination of direct reverse-phase HPLC analysis, substitution of butyryl for acetyl coenzyme A substrate, automated sample loading and continuous on-line radioactivity detection significantly improves the assay. This technique eliminates the need for organic extraction; improves chromatographic resolution of radioactive substrate and products; provides low background with high sensitivity, linearity, and precision; and yields rapid, quantitative results, even at low levels of cellular CAT expression.
Zajac
J D
JD
Endocrine Unit, Massachusetts General Hospital, Boston 02114.
Gerardi
J M
JM
Kearns
A K
AK
Bringhurst
F R
FR
D000818:Animals; D001322:Autoanalysis; D002460:Cell Line; D015500:Chloramphenicol O-Acetyltransferase / Q000032:analysis* / Q000235:genetics; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D014162:Transfection
D015500:Chloramphenicol O-Acetyltransferase