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.
2022-01-20 19:17:10