Dear Yong, I've used both BME and DTT (as well as CoA, Cys, or GSH, etc.) to terminate/exhaust covalent chemistry on Cys residues. It's relatively speaking 'a piece of cake' however one has to keep in mind that if the particular covalent reagent is very highly specific towards a Cys residue in a protein's targeted microenvironment, that reagent may not be reactive enough to effectively quench with BME or DTT. In other terms, the 'warhead' reactivity may be dialed down so much that it does not react readily with free thiols. You may want to confirm your outcome somehow (e.g. by small molecule MS).
It's not like this reaction is hard to set up, or requires massive optimization of conditions - generally speaking, as long as you have 'much more' of DTT or BME than you have of the covalent agent, given some time the reaction will go to completion. All the best, Artem https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4410814/ "*Excess alkylating compound was quenched with 1 mM DTT*" https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6310217/ "*...useful post-reaction to quench any remaining maleimide after achieving the desired conjugation levels.*" https://pubmed.ncbi.nlm.nih.gov/26387744/ "*...N-acetyl cysteine (21 μL of 100 mM stock in water, 2.2 μmol, 40 eq) was added to quench unreacted maleimide.*" - Cosmic Cats approve of this message On Tue, Aug 27, 2024 at 3:17 PM Yong Tang <liutan...@gmail.com> wrote: > Dear all, happy summer! > > I searched in many ways but could not find relevant references easily > so I'm hoping you could help. > > For TSA assays on a Cys-containing protein (drug target) that has just > gone through a covalent adduction reaction with a compound carrying a > Michael acceptor covalent warhead, we would like to stop the further > progression of the covalent adduction before subjecting the reaction > mixture to TSA analysis. It is my understanding that people usually use DTT > to deplete the excess covalent compound - do you happen to come across such > a scientific reference? > > Thank you so much in advance, -yong > > > ------------------------------ > > To unsubscribe from the CCP4BB list, click the following link: > https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1 > ######################################################################## To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1 This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a mailing list hosted by www.jiscmail.ac.uk, terms & conditions are available at https://www.jiscmail.ac.uk/policyandsecurity/
