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break disulfide bonds to study fluorescence

Biophysics Fluorescence Quenching Break disulfide bonds Reducing agent Protein

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#1 Mulligan

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Posted 27 April 2013 - 08:09 AM

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.

#2 HOYAJM

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Posted 06 May 2013 - 09:25 AM

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

#3 labtastic

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Posted 14 May 2013 - 10:59 AM

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. Posted Image

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.






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