期刊
NATURE PROTOCOLS
卷 4, 期 9, 页码 1363-1371出版社
NATURE PUBLISHING GROUP
DOI: 10.1038/nprot.2009.128
关键词
-
资金
- Cancer research UK program grant
- Ramon y Cajal Program
- Ministerio de Educacion y Ciencia [BFU 2005-04091]
- Fondo de Investigaciones Sanitarias
- CIBERhed
The small ubiquitin-like modifiers (SUMOs) are posttranslationally conjugated to eukaryotic cellular proteins with generally unpredictable consequences. SUMO substrates are found in many cellular systems, and functional analysis has revealed that substrate SUMOylation often has an important role in their regulation. Here we describe a cell-based protocol which can be used to detect the SUMOylation of a protein that relies on the enrichment of SUMO conjugates by purification of 6His-SUMO under denaturing conditions, followed by western blotting for the protein of interest. By purifying under denaturing conditions this method not only reduces the risk of false-positive identifications by non-covalent interactions, but also preserves SUMO-substrate conjugates by inhibiting SUMO-specific proteases-two caveats that may complicate other less stringent purification methods. In preliminary form, this protocol takes 4-5 d to perform, and it can be further elaborated to provide a multi-angled approach to investigate protein conjugation by SUMO.
作者
我是这篇论文的作者
点击您的名字以认领此论文并将其添加到您的个人资料中。
推荐
暂无数据