I am running a reaction to quantify the thiol groups of my chitosan. However, when I use Ellman's reagent and add it to my standard and samples there is no colour change. My buffer is 0.1M sodium phosphate pH 8.0 with 1mM of EDTA, and all procedures were followed as provided on the Thermo Fischer website for the DTNB. If anyone can help, it would be much appreciated.
This is an extremely robust assay and very rarely fails. Your buffer is correct and a negative result signifies one of three things; you have no thiols, or the conditions are not correct to develop the colour, or you have an error in the preparation of the DTNB and/or standards. The first check I would make is qualitative - add 1ul of mercaptoethanol stock to 200ul of 1x dtnb reagent. If you don’t see a strong yellow colour instantly you definitely have an issue with your DTNB reagent. If you do see a strong colour, you have an issue with your standards. Which thiol are you using as the standard and what are you dissolving and diluting it in? The thing to watch out for here is pH. The standard must not depress the pH of the DTNB reagent. Dilution errors obviously also need to be ruled out.
Nick Gee It seems to have worked once before. The thiol I'm using as standard is cysteine hydrochloride monohydrate. I am dissolving the cysteine in the reaction buffer which is (0.1M sodium phosphate at pH 8.0 with 1mM of EDTA).
Chitosan, at least in its pure state, does not contain thiols. It is a mixed polymer of N-acetylglucosamine and glucosamine. Any thiols you detect are impurities.
Moreover, thiols are prone to oxidation in air, so it is unlikely that thiols present as impurities will be in the reduced state that will allow them to be measured, unless care has been taken to prevent their oxidation.
If you are not able to detect freshly dissolved cysteine with Ellman's reagent, there is something badly wrong. This is a very robust detection method, as Nick Gee said. You should be able to detect cysteine at concentrations in the 10 µM range by the absorbance change at about 412 nm.
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