Isolation of In Vivo SUMOylated Chromatin-Bound Proteins
Isolation of In Vivo SUMOylated Chromatin-Bound Proteins
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DOI：
10.1007/978-1-4939-6358-4_15
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发表时间：
2016-01-01
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影响因子：
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通讯作者：
Bawa-Khalfe, Tasneem
中科院分区：
文献类型：
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作者：
Bawa-Khalfe, Tasneem
SUMO 翻译后修饰指导基因转录和表观遗传编程以支持正常细胞功能。 SUMO 修饰的动态性质使得鉴定内源蛋白质底物变得困难。染色质结合 SUMO 靶标的分离极具挑战性，因为传统的免疫沉淀测定在浓缩该蛋白质群体方面效率低下。本章描述了在培养细胞中有效沉淀 SUMO 化异染色质蛋白 1 α 的染色质相关组分的方案。还演示和讨论了在染色质处富集内源 SUMO 底物的技术。这种方法可以适用于评估其他体内系统中染色质结合的 SUMO 靶标。
SUMO posttranslational modification directs gene transcription and epigenetic programming to support normal cell function. The dynamic nature of SUMO-modification makes it difficult to identify endogenous protein substrates. Isolation of chromatin-bound SUMO targets is exceptionally challenging, as conventional immunoprecipitation assays are inefficient at concentrating this protein population. This chapter describes a protocol that effectively precipitates chromatin-associated fractions of SUMOylated heterochromatin protein 1 alpha in cultured cells. Techniques to enrich endogenous SUMO substrates at the chromatin are also demonstrated and discussed. This approach could be adapted to evaluate chromatin-bound SUMO targets in additional in vivo systems.