简单高效地鉴定染色质修饰复合物并表征复合物组成。

Simple and efficient identification of chromatin modifying complexes and characterization of complex composition.

作者信息

Lee Jeong-Heon, Skalnik David

机构信息

Department of Pediatrics, Wells Center for Pediatric Research, Indiana University School of Medicine, Indianapolis, IN, USA.

出版信息

Methods Mol Biol. 2013;977:289-98. doi: 10.1007/978-1-62703-284-1_23.

DOI:10.1007/978-1-62703-284-1_23

PMID:23436371

Abstract

Affinity purification and mass spectrometry analysis have been used to identify and characterize protein complexes. Wdr82-associated chromatin modifying complexes were purified by single-step FLAG affinity purification from human cells induced to express FLAG-tagged Wdr82. Purified proteins were analyzed by SDS-PAGE and specific protein bands were identified by mass spectrometry. Subsequently, purified proteins were fractionated on sucrose gradient equilibrium centrifugation to determine overall composition of each identified complex. We describe here simple and efficient approaches for the identification of chromatin modifying complexes and subsequent characterization of complex composition.

摘要

亲和纯化和质谱分析已被用于鉴定和表征蛋白质复合物。通过单步FLAG亲和纯化从诱导表达FLAG标签的Wdr82的人细胞中纯化与Wdr82相关的染色质修饰复合物。通过SDS-PAGE分析纯化的蛋白质，并通过质谱鉴定特定的蛋白条带。随后，将纯化的蛋白质在蔗糖梯度平衡离心中分级分离，以确定每个鉴定出的复合物的整体组成。我们在此描述了用于鉴定染色质修饰复合物以及随后表征复合物组成的简单有效的方法。