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Methods Mol Biol. 2012;876:163-72. doi: 10.1007/978-1-61779-809-2_13.

In vitro protein ubiquitination assay.

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State Key Laboratory of Plant Genomics, National Center for Plant Gene Research, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, China.


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.

[Indexed for MEDLINE]

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