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一种通过体内泛素化检测和CCK-8检测来检测蛋白质泛素化及泛素化激活的优化方案。

An optimized protocol to detect protein ubiquitination and activation by ubiquitination assay in vivo and CCK-8 assay.

作者信息

Lin Xiao-Tong, Zhang Jie, Xie Chuan-Ming

机构信息

Key Laboratory of Hepatobiliary and Pancreatic Surgery, Institute of Hepatobiliary Surgery, Southwest Hospital, Third Military Medical University (Army Medical University), Chongqing, China.

Key Laboratory of Hepatobiliary and Pancreatic Surgery, Institute of Hepatobiliary Surgery, Southwest Hospital, Third Military Medical University (Army Medical University), Chongqing, China.

出版信息

STAR Protoc. 2023 Mar 24;4(2):102199. doi: 10.1016/j.xpro.2023.102199.

DOI:10.1016/j.xpro.2023.102199
PMID:36964909
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC10050764/
Abstract

E3 ubiquitin ligases play a role in protein degradation, cellular localization, and activation, and their dysregulation is associated with human diseases. Here, we present a protocol to detect IGF2BP1 ubiquitination and activation by an E3 ubiquitin ligase FBXO45. We describe steps for preparing cells and transfecting plasmids. We detail the use of western blot to detect IGF2BP1 ubiquitination and a Cell Counting Kit-8 (CCK-8) assay to detect IGF2BP1 activation. This protocol is applicable to other proteins of interest. For complete details on the use and execution of this protocol, please refer to Lin et al. (2021)..

摘要

E3泛素连接酶在蛋白质降解、细胞定位和激活过程中发挥作用，其失调与人类疾病相关。在此，我们展示了一种检测E3泛素连接酶FBXO45介导的IGF2BP1泛素化和激活的方法。我们描述了细胞制备和质粒转染的步骤。我们详细说明了使用蛋白质印迹法检测IGF2BP1泛素化以及使用细胞计数试剂盒-8（CCK-8）检测IGF2BP1激活的方法。该方法适用于其他感兴趣的蛋白质。有关此方法的使用和执行的完整详细信息，请参考Lin等人（2021年）的文献。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/6555/10050764/0d09c7456f79/gr3.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/6555/10050764/f86bc4deca01/fx1.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/6555/10050764/2cf0f0fb55de/gr1.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/6555/10050764/d7d913ebf67b/gr2.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/6555/10050764/0d09c7456f79/gr3.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/6555/10050764/f86bc4deca01/fx1.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/6555/10050764/2cf0f0fb55de/gr1.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/6555/10050764/d7d913ebf67b/gr2.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/6555/10050764/0d09c7456f79/gr3.jpg

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