Another option is DTNB (aka Ellman's Reagent). This compound reacts with free thiols and produces a yellow color. The reaction is stoichiometric, so if you have access to a very basic spectrophotometer and know your protein concentration you can quantify the free thiols quite easily.
Basic pH doesn't break disulfides, if anything it stabilizes them. I would be more concerned that they are incorrectly formed as described by Herman. Mike ----- Original Message ----- From: "D Bonsor" <dbon...@ihv.umaryland.edu> To: CCP4BB@JISCMAIL.AC.UK Sent: Friday, August 19, 2011 6:21:41 AM GMT -08:00 US/Canada Pacific Subject: Re: [ccp4bb] Off topic_protein degradation. If you are unsure about whether the disulfides have formed treat a small amount of protein with N-ethylmaleimide. If the disulfides have not formed, when you perform mass spec on the protein you should see an increase of mass of 125Da for each exposed cysteine. -- Michael C. Thompson Graduate Student Biochemistry & Molecular Biology Division Department of Chemistry & Biochemistry University of California, Los Angeles mi...@chem.ucla.edu
