鉴定cGKI底物的方法。

Methods for identification of cGKI substrates.

作者信息

Salb Katharina, Schlossmann Jens

机构信息

Pharmakologie und Toxikologie, Institut für Pharmazie, Universität Regensburg, Regensburg, Germany.

出版信息

Methods Mol Biol. 2013;1020:147-62. doi: 10.1007/978-1-62703-459-3_9.

DOI:10.1007/978-1-62703-459-3_9

PMID:23709031

Abstract

The cGMP-dependent protein kinases (cGK), which belong to the family of serine/threonine kinases, exhibit their diverse functions in cells through interaction with a variety of substrate proteins. Several substrates were identified and the interactions studied using different methods inter alia co-immunoprecipitation (Co-IP) and cGMP-agarose affinity purification. In the following chapter, we will describe the preparation of cell or tissue lysates, the procedures of cGMP-agarose affinity purification and co-immunoprecipitation, and finally the separation and analysis of the protein complexes by SDS-PAGE or mass spectrometry.

摘要

环磷酸鸟苷（cGMP）依赖性蛋白激酶（cGK）属于丝氨酸/苏氨酸激酶家族，通过与多种底物蛋白相互作用在细胞中发挥多种功能。已鉴定出几种底物，并使用多种方法研究了相互作用，尤其是免疫共沉淀（Co-IP）和cGMP-琼脂糖亲和纯化。在接下来的章节中，我们将描述细胞或组织裂解物的制备、cGMP-琼脂糖亲和纯化和免疫共沉淀的步骤，最后通过SDS-PAGE或质谱对蛋白质复合物进行分离和分析。