Abstract
Ubiquitin can be added to substrate protein as a protein tag by the concerted actions of ubiquitin activating enzyme (E1), ubiquitin conjugating enzyme (E2) and ubiquitin protein ligase (E3). At the present of E1 and ubiquitin, E2 activity can be determined by the thio-ester formation. The E3 activity of a putative protein as well as the E2/E3 or E3/substrate specificities also can be explored by in vitro ubiquitination assay. The result can be detected by western blot with certain antibody. Purified proteins expressed from bacterial system are always used in this assay.
Keywords: Ubiquitination, In vitro, Ubiquitin ligase, Ubiquitin conjugating enzyme
Materials and Reagents
Equipment
Procedure
Recipes
Acknowledgments
This protocol was developed from the following published paper: Zhao et al. (2013). This work was supported by grants from the National Basic Research Program of China (973 Program) (2011CB915402) and the National Science Foundation of China (CNSF 31030047/90717006). Zhao QZ is supported by National Science Foundation of China grant CNSF 31200907.
References
If you have any questions/comments about this protocol, you are highly recommended to post here. We will invite the authors of this protocol as well as some of its users to address your questions/comments. To make it easier for them to help you, you are encouraged to post your data including images for the troubleshooting.