DTNBbased

DTNBbased refers to methods and reagents that rely on Ellman's reagent, 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), to detect and quantify thiol groups in samples. In a typical DTNBbased reaction, a free thiol (R-SH) reduces DTNB to produce one molecule of 5-thio-2-nitrobenzoic acid (TNB−), the yellow chromophore, along with a mixed disulfide (R-S-S-TNB). The formation of TNB− yields an absorbance at 412 nm, which can be quantified using a standard curve and Beer-Lambert law. The method is commonly performed under mildly alkaline conditions and can be adapted for cuvettes or microplate formats.

DTNBbased assays are widely used to measure thiol content in small molecules, peptides, and proteins, and to quantify antioxidants such as reduced glutathione or free cysteine residues. For protein samples, protocols often require preparation steps to expose or liberate thiols, and sometimes a reducing step to determine total thiols versus free thiols. Standards of known thiol concentration are used to calibrate the assay, enabling calculation of thiol concentrations in unknown samples.

Advantages of DTNBbased approaches include simplicity, speed, and relatively low cost, along with compatibility with many sample types and plate readers. Limitations include interference from substances that absorb at 412 nm or react with DTNB, reduced accuracy with very turbid or colored samples, and the need to consider disulfide-linked thiols separately from free thiols. DTNBbased methods are sometimes contrasted with alternative thiol-detection approaches that offer greater specificity or sensitivity under particular conditions. Safety considerations include handling DTNB with appropriate precautions due to its reactive and potentially hazardous nature.