Zajac J D, Gerardi J M, Kearns A K, Bringhurst F R
Endocrine Unit, Massachusetts General Hospital, Boston 02114.
DNA. 1988 Sep;7(7):509-13. doi: 10.1089/dna.1.1988.7.509.
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.
氯霉素乙酰转移酶（CAT）在真核基因表达研究中被广泛用作报告元件。我们报告了一种测量CAT活性的新技术，该技术相比其衍生而来的现有方法具有若干优势。直接反相高效液相色谱分析、用丁酰辅酶A替代乙酰辅酶A底物、自动进样以及连续在线放射性检测相结合，显著改进了该测定方法。这项技术无需进行有机萃取；提高了放射性底物和产物的色谱分辨率；具有低背景、高灵敏度、线性度和精密度；即使在细胞CAT表达水平较低时也能快速得出定量结果。
