In vitro Ubiquitin Dimer Formation Assay   

Edited by
Yanjie Li
Reviewed by
Anonymous reviewer
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In this protocol

Original research article

A brief version of this protocol appeared in:
Journal of Experimental Botany
May 2016


The process of protein ubiquitination typically consists of three sequential steps to add an ubiquitin (Ub) or Ub chain to a substrate protein, requiring three different enzymes, ubiquitin activating enzyme (E1), ubiquitin conjugating enzyme (E2), and ubiquitin protein ligase (E3). Most E2s possess the classical E2 activity in forming E2-Ub complex through a thioester linkage, in presence of an E1 and Ub. Additionally, some E2s have the ability of catalyzing the formation of free Ub dimer. Such activity indicates an important role of these E2s in ubiquitination pathway. Thus, we developed an in vitro Ub dimer formation assay to determine the activity of certain E2s. Moreover, by using Ub mutants, in which different lysine residues are mutated, the specific linkage of dimer can also be determined.

Keywords: Ubiquitination, Ubiquitin dimer formation, E2, Arabidopsis UBC22, K11 linkage


The existing protocols for E2 conjugation initiation assay (without adding E3 and substrate) aim to detect the thioester linkage (E2-S-Ub). Our method focuses on the E2 activity of catalyzing free Ub dimer formation (Ub-Ub). It provides a convenient way to detect an important biochemical feature of E2 in different species. Further, the specific linkage of dimer can be determined by using different Ub mutants.

Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
How to cite: Wang, S., Cao, L. and Wang, H. (2017). In vitro Ubiquitin Dimer Formation Assay. Bio-protocol 7(1): e2082. DOI: 10.21769/BioProtoc.2082.

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