In vitro Auto- and Substrate-Ubiquitination Assays
In vitro Auto- and Substrate-Ubiquitination Assays
复制标题
体外自动和底物泛素化试验
DOI：
10.21769/bioprotoc.4368
复制
发表时间：
2022-04-05
期刊：
影响因子：
0.8
通讯作者：
Yoon, Gyeong Mee
中科院分区：
文献类型：
--
作者：
Park, Hye Lin;Lee, Han Yong;Yoon, Gyeong Mee
细胞蛋白质组稳态的精确调控对植物的正常生长发育至关重要。它还允许植物通过及时调节其生化和生理方面来应对各种环境胁迫。细胞蛋白的泛素化是维持细胞蛋白稳态的主要蛋白质降解途径之一，泛素E3连接酶作为泛素连接酶复合物的组成部分，通过底物特异性相互作用在靶蛋白的选择性降解中发挥重要作用。因此，了解E3连接酶的作用及其底物调控可以揭示其特定的细胞和生理功能。在这里，我们提供了自体和底物泛素化分析的方案，利用体外纯化的E3泛素连接酶蛋白和免疫沉淀的组合。
The precise regulation of the homeostasis of the cellular proteome is critical for the appropriate growth and development of plants. It also allows the plants to respond to various environmental stresses, by modulating their biochemical and physiological aspects in a timely manner. Ubiquitination of cellular proteins is one of the major protein degradation routes for maintaining cellular protein homeostasis, and ubiquitin E3 ligases, components of ubiquitin ligase complexes, play an important role in the selective degradation of target proteins via substrate-specific interactions. Thus, understanding the role of E3 ligases and their substrate regulation uncovers their specific cellular and physiological functions. Here, we provide protocols for auto-and substrate-ubiquitination analyses that utilize the combination of in vitro purified E3 ubiquitin ligase proteins and immunoprecipitation.