2 replies to this topic

[Mulligan]

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.

[HOYAJM]

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

[labtastic]

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.

Edited by labtastic, 14 May 2013 - 11:02 AM.