使用非变性聚丙烯酰胺凝胶电泳分析细胞提取物中的酪氨酸激酶活性。

Analysis of tyrosine kinase activity in cell extracts using nondenaturing polyacrylamide gel electrophoresis.

作者信息

Glazer R I, Yu G, Knode M C

机构信息

Laboratory of Biological Chemistry, National Cancer Institute, Bethesda, Maryland 20892.

出版信息

Anal Biochem. 1987 Jul;164(1):214-20. doi: 10.1016/0003-2697(87)90388-5.

DOI:10.1016/0003-2697(87)90388-5

PMID:2445220

Abstract

A general procedure for detecting tyrosine kinase activity in crude or purified preparations using nondenaturing gel electrophoresis is presented. Samples are resolved by electrophoresis in minigels which are then incubated in an assay mixture containing [gamma-32P]ATP, poly(glutamic acid, tyrosine)4:1, and cofactors. Subsequently, gels are fixed and washed in trichloroacetic acid-pyrophosphate, dried, and analyzed by autoradiography or liquid scintillation counting. The procedure is simple and fast and allows analysis of different molecular weight forms of tyrosine kinase under linear kinetics at 37 degrees C without interference from phosphatases and proteases.

摘要

本文介绍了一种使用非变性凝胶电泳检测粗提物或纯化物中酪氨酸激酶活性的通用方法。样品在微型凝胶中进行电泳分离，然后在含有[γ-32P]ATP、聚（谷氨酸，酪氨酸）4:1和辅因子的测定混合物中孵育。随后，凝胶进行固定，在三氯乙酸-焦磷酸中洗涤，干燥，并通过放射自显影或液体闪烁计数进行分析。该方法简单快速，可在37℃线性动力学条件下分析不同分子量形式的酪氨酸激酶，且不受磷酸酶和蛋白酶的干扰。