Thiols are organosulfur compounds containing a thiol group or sulfhydryl group. A thiol group is the sulfur analogue of a hydroxyl group. Due to its high reactivity, thiol plays an important role in biomolecules. For example, cysteine residues contain thiol groups and can form cystine which has a disulfide bond to maintain the specific structure and stability of a protein. Many enzymes and cofactors such as coenzyme A rely on a thiol group as its key reactive group. In addition, thiols are easily oxidized, and glutathione is an important antioxidant in cells. These properties make changes in thiol levels a good indicator of diseases, oxidative stress, and enzyme activity.

Free thiols react with 5,5′-dithiobis-(2-nitrobenzoic acid), also known as DTNB or Ellman’s reagent, to release 2-nitro-5-thiobenzoate (TNB⁻) as the below figure shows. TNB⁻ ionizes to the TNB²⁻ dianion at neutral and alkaline pH in aqueous solvent, which shows yellow color. Because the formation of TNB⁻ is proportional to thiol, this reaction can be used for quantifying the amount of thiol.

RayBio^® Total Thiol Quantification Assay (Ellman’s test) Kit is designed based on the Ellman’s reaction and optimized for the high-throughput microtiter form. It can be used to easily and quickly measure the amount of total free thiol in a given sample with a range of 0-40 nmole per well.

• DTNB reagent
• >Assay Diluent F
• L-cysteine standard
• 96 well microplate

• Microplate reader capable of measuring absorbance at 412 nm
• Precision pipettes to deliver 2 µl to 1 ml volumes
• Serological Pipettes (25 ml)
• Microcentrifuge tubes to prepare sample dilutions
• 50 ml conical tubes for preparing reagent working solution

1. In the microplate, add 25 μl of each standard or sample to wells in duplicate or triplicate.
2. Add 0.25 ml of the DNTB working solution into each well.
3. Read the absorbance at 412 nm (A412) by plate reader within 10 minutes. No incubation is needed.