A Nonchromatographic Assay for Expression of the Chloramphenicol Acetyltransferase Gene in Eucaryotic Cells

Overview

Journal Anal Biochem

Publisher Elsevier

Specialty Biochemistry

Date 1986 Jul 1

PMID 3461723

Citations 128

Authors

M J Sleigh

Affiliations

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Abstract

A rapid procedure is described for assaying chloramphenicol acetyltransferase (CAT) enzyme activity following transfection of the CAT gene into eucaryotic cells. CAT enzyme activity in cell extracts catalyzes the transfer of [14C]acetyl groups from labeled acetyl coenzyme A to unlabeled chloramphenicol. Labeled reaction product is quantitated by liquid scintillation counting after extraction into ethyl acetate. The method is valid for use with transfected cell extracts only if the extracts are first heated to 65 degrees C to remove a factor which degrades acetyl coenzyme A. The revised procedure offers considerable advantages in speed and ease of performance over the chromatographic assay in current use.

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