In vitro protein ubiquitination assay

Methods in Molecular Biology
Qingzhen ZhaoQi Xie

Abstract

Ubiquitination is one of the most important posttranslational modifications in all eukaryote organisms. Ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzyme (E2), and ubiquitin ligase (E3) are the three key enzymes in this process. To detect the specificity between E2 and E3 or enzyme-substrate relationship between E3 and a substrate protein, ubiquitination activity needs to be determined. This protocol provides a convenient and efficient in vitro assay for DTT-sensitive thioester formation of E2s and Ring/U-box-type E3s, and E3-mediated substrate ubiquitination. E2/E3 specificities can also be investigated quickly by using this system. This method can be applied to ubiquitination assays of proteins from any eukaryotic organisms.

Citations

Feb 2, 2018·Applied Microbiology and Biotechnology·Arshad Ahmed PadhiarMintao Zhong
Jun 12, 2018·Bio-protocol·Xian FuJulie A Maupin-Furlow
Apr 26, 2020·Protein Expression and Purification·A Katherine Hatstat, Dewey G McCafferty
Mar 5, 2021·Proceedings of the National Academy of Sciences of the United States of America·Yachun LinYuanchao Wang
Oct 1, 2021·The Biochemical Journal·Samuel R WitusRachel E Klevit
Oct 14, 2021·Bioscience, Biotechnology, and Biochemistry·Soomi KimHongtae Kim

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