蛋白质 Pull-down 实验检测细菌 RNA 降解体的组成。

Protein Pulldown Assays to Monitor the Composition of the Bacterial RNA Degradosome.

机构信息

Department of Biochemistry, University of Cambridge, Cambridge, UK.

Departamento de Microbiologia, Instituto de Ciências Biomédicas, Universidade de São Paulo, São Paulo, SP, Brazil.

出版信息

Methods Mol Biol. 2021;2209:425-432. doi: 10.1007/978-1-0716-0935-4_26.

DOI:10.1007/978-1-0716-0935-4_26

PMID:33201484

Abstract

The method of co-immunoprecipitation (co-IP or pulldown) enables the identification of proteins interacting in macromolecular assemblies, through the purification of a key protein by affinity chromatography using specific antibodies immobilized on a matrix. The advantages of using epitope-tagged proteins include the ability to use commercially available antibodies for affinity purifications, and typically they do not disrupt the structure of the protein complexes. Here we describe the utilization of an epitope-tagged version of Caulobacter crescentus RNase E in order to determine the composition of the RNA degradosome under different growth conditions. Several proteins that interact with the RNA degradosome were identified.

摘要

免疫共沉淀（co-IP 或下拉）方法可通过使用固定在基质上的特异性抗体对关键蛋白质进行亲和层析，从而鉴定大分子组装体中相互作用的蛋白质。使用表位标记蛋白的优点包括能够使用商业上可获得的抗体进行亲和纯化，并且通常它们不会破坏蛋白质复合物的结构。在这里，我们描述了使用新月柄杆菌 RNase E 的表位标记版本来确定不同生长条件下 RNA 降解体的组成。鉴定出了几种与 RNA 降解体相互作用的蛋白质。