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
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Thank you for your question! Just as your said, if the 20 x reaction buffer contains DTT, it is not good for the thiolester bond detection in Procedure A. If the thioester bond formed by E2 and ubiquitin is strong enough, we can also get the DTT-sensitive bond since the DTT concentration in the working 1x reaction buffer is much lower. But obviously it is better if there is not DTT in the reaction buffer and it's important for the weak binding force between E2 and ubiquitin. The components of 20 x reaction buffer in the recipies are suitble for Procedure B and C. For Procedure A, DTT should be removed from the 20 x reaction buffer and other components remain unchanged. I should have noticed this before. I will revise it in the protocol and thank you very much for your reminding.
thank you very much, I'd like to ask you again,What is the total volume of 20x reaction buffer?
The total volume of 20* buffer is according to your own needs. I often prepare 1-2 ml 20* buffer each time and then aliquot to 20 ul/tube,stored at -20℃.