| Bioactivity | DTNB (Ellman’s Reagent) is a chemical used to quantify the number or concentration of thiol groups[1]. | ||||||||||||
| Invitro | DTNB reacts with the free sulfhydryl side chain of cysteine to form an S-S bond between the protein and a thionitrobenzoic acid (TNB) residue[1]. The main advantage of DTNB over alternative reagents (e.g., N-ethylmaleimide or iodoacetamide) is in the selectivity of this reagent and in the ability to follow the course of the reaction spectrophotometrically[1].Modification of Enzyme with DTNB:Modification of the SH groups of the enzyme is carried out by reacting 10 μL of 10 mM DTNB solution (about a 20-fold molar excess) at room temperature with 0.6 mL of enzyme solution (0.807 mg/mL) in 80 mM phosphate buffer, pH 8.0, containing 1 mM EDTA, which has been dialyzed previously against the same buffer solution for 24 h. The number of SH groups is estimated from the increase of absorbance at 410 nm using a molar extinction coefficient of 13,600 M-1cm-1 for thionitrobenzoate ions liberated. | ||||||||||||
| Name | DTNB | ||||||||||||
| CAS | 69-78-3 | ||||||||||||
| Formula | C14H8N2O8S2 | ||||||||||||
| Molar Mass | 396.35 | ||||||||||||
| Appearance | Solid | ||||||||||||
| Transport | Room temperature in continental US; may vary elsewhere. | ||||||||||||
| Storage |
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| Reference | [1]. T Nagaoka, et al. DTNB modification of SH groups of isocitrate dehydrogenase from Bacillus stearothermophilus purified by affinity chromatography. J Biochem. 1977 Jan;81(1):71-8. |