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.