In vitro Di-ubiquitin Formation Assay and E3 Cooperation Assay

Rebecca J. Burge, Katie H. Jameson, Anthony J. Wilkinson, Jeremy C. Mottram*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review


Ubiquitination is a post-translational modification conserved across eukaryotic species. It contributes to a variety of regulatory pathways, including proteasomal degradation, DNA repair, and cellular differentiation. The ubiquitination of substrate proteins typically requires three ubiquitination enzymes: a ubiquitin-activating E1, a ubiquitin-conjugating E2, and an E3 ubiquitin ligase. Cooperation between E2s and E3s is required for substrate ubiquitination, but some ubiquitin-conjugating E2s are also able to catalyze by themselves the formation of free di-ubiquitin, independently or in cooperation with a ubiquitin E2 variant. Here, we describe a method for assessing (i) di-ubiquitin formation by an E1 together with an E2 and an E2 variant, and (ii) the cooperation of an E3 with an E1 and E2 (with or without the E2 variant). Reaction products are assessed using western blotting with one of two antibodies: the first detects all ubiquitin conjugates, while the second specifically recognizes K63-linked ubiquitin. This allows unambiguous identification of ubiquitinated species and assessment of whether K63 linkages are present. We have developed these methods for studying ubiquitination proteins of Leishmania mexicana, specifically the activities of the E2, UBC2, and the ubiquitin E2 variant UEV1, but we anticipate the assays to be applicable to other ubiquitination systems with UBC2/UEV1 orthologues.

Original languageEnglish
Article numbere4547
Number of pages8
Issue number21
Publication statusPublished - 5 Nov 2022

Bibliographical note

Funding Information:
This protocol was adapted from the research published in Burge et al. (2020). We would like to thank Ubiquigent for providing a 3 month internship for RB, during which this protocol was developed. This work was supported by a Medical Research Council Studentship to RB (MRC MR/N018230/1) and the Wellcome Trust to JCM (200807/Z/16/Z).

Publisher Copyright:
Copyright: © 2022 The Authors; exclusive licensee Bio-protocol LLC.


  • Chain formation
  • E2 conjugating
  • E3 ligase
  • Ubiquitin
  • Ubiquitination

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