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break disulfide bonds to study fluorescence - (Apr/27/2013 )

Hi everyone! im new in this forum.

I need to resolve a problem with a denatured a protein with 2-mercaptoethanol and 8 M urea . I need to study quenching on Trp and sadly 2-mercaptoethanol its a quencher. So i need to obtain a denature protein without disulfide bonds in 8 M urea but without 2-mercaptoethanol. I tought of making an ultrafiltration by keeping the volume constant and the solution of 8 M urea. However, my concern is if I change the reducing media the protien will oxidise the Cys again.
I tought use glutathione and find if is a quencher too but i first want to try taking off the 2-mercaptoethanol.

I'd very much appreciate your comments

Thank you very much.


use DTT instead of 2-mercaptoethanol. DTT will break the disulfide bonds just as well.


If glutathione and beta-mercaptoethanol are Trp fluorescence quechers, I suspect DTT will be also because it's probably the sulfur that is doing the quenching.

Instead, use TCEP. It's a commonly used cysteine reducing agent but is different from DTT, BME and glutathione in that it is not sulfur based.


hai ,, can any body tell me what is the mechanism of tryptophan fluorescence quenching by BME ??