Sensitive Radiometric Assay for Chloramphenicol Acetyltransferase Using Automated HPLC

Abstract

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 level of cellular CAT expression.