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. © 2012 Springer Science+Business Media, LLC.
CITATION STYLE
Zhao, Q., Liu, L., & Xie, Q. (2012). In vitro protein ubiquitination assay. Methods in Molecular Biology, 876, 163–172. https://doi.org/10.1007/978-1-61779-809-2_13
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