Hi, I'm familiar with the reaction, but that reagent is generally used to estimate free thiols, not disulfides.

Yes I know. First we determine free thiols, then we manage to 'break' existent S-S bonds and determine again the free thiols, the difference will allow us to estimate the disulfide bonds

Yes, you can do it that way.

Alternatively, you can block the free thiols with maleimide and then reduce the protein and use DTNB to measure the freed thiols. Reducing agents, like DTT and b-mercaptoethanol, do not cleave the thiol-maleimide bond, but they will cleave the thiol-DTNB bond.

Yes, DTNB with a suitable buffer is an easy way to determine the amount of free SH. I would recommend to consider the molar adsorption coefficients by Riddles et al. (1) and Eyer et al.(2) If you are interested have a look at our paper in which we applied Ellman to determine the surface concentration of SH on flat silicon and glass substrates. (3)

(1) Riddles, P.; Blakeley, R.; Zerner, B. Method Enzymol. 1983, 91, 49−60.

(2) Eyer, P.; Worek, F.; Kiderlen, D.; Sinko, G.; Stuglin, A.; Simeon- Rudolf, V.; Reiner, E. Anal. Biochem. 2003, 312, 224−227.

(3) Kuttner, C.; Tebbe, M.; Schlaad, H.; Burgert, I.; Fery, A. Acs Appl Mater Inter 2012, 4, 3485–3492.

We have used it as part of a system for detection inhibition od acetylcholinesterase. Worked fine.

Hi, you can also detect sulfite with DTNB. We even applied it in an in vitro enzyme assay.

The Elman reagent is a dimmer of 3-carboxy-4-nitro-thiophenol. Can be prepared from 2-nitro-5-chlorobenzoic acid. It is sold by Sigma Aldrich.