DTNB (Ellman's Reagent)

DTNB Usage Instructions ^[1]:

Standard solution (10 mM): During each step, it is essential to avoid exposure to light. Start by weighing out 0.198 grams of DTNB and prepare a 50 mL solution in 50 mM Na₂HPO₄ (pH 7.0). Store this solution in a brown or amber-colored bottle, and keep it in a cool(4℃), dark place for later use.

Protocol for Measurement of Total GSH in Caenorhabditis elegans using DTNB ^[2]:

1. Load the 96-well plate with 20 μl KPE buffer for blank, 20 μl GSH standard, or 20 μl sample into each well. Blank, standards and samples should be loaded in duplicate.
2. Mix DTNB and GR solutions together (1:1). Add 120 μl to each well using a multichannel pipettor.
3. Wait 30 sec and then add 60 μl of NADPH solution to each well using a multichannel pipettor.
4. Immediately read absorbance at 412 nm every 30 sec for 2 min.
5. Calculate the rate of 2-nitro-5-thiobenoic acid formation (i.e., change in absorbance per min) for standards and samples.
6. Generate a standard curve by plotting standard concentration vs. rate of 2-nitro-5thiobenoic acid formation.
7. Determine the GSH concentration in sample wells by using linear regression for the standard curve.
8. Quantify protein content of the GSH extract using the remainder of the extract.

DTNB solution: 2 mg DTNB;3 ml KPE buffer; Prepare fresh each time, store on ice, wrapped in aluminum foil.

KPE buffer (pH 7.5): Solution A: 6.8 g KH₂PO₄ plus 500 ml sterile H₂O, store at 4℃；Solution B: 11.4 g K₂HPO₄-3H₂O in 500 ml sterile H₂O, store at 4℃；Add 16 ml solution A to 84 ml solution B; add 0.327 g EDTA Store at 4℃, up to 1 month.

Glutathione reductase (GR) solution: 40 μl GR (250 U/ml);3 ml KPE buffer; Prepare fresh each time, store on ice.

GSH standards: Stock 2 mM GSH: 6.14 mg GSH in 10 ml KPE buffer, store as 1ml aliquots at -20℃ up to 1 month. Serially dilute starting at 40 μM GSH (20 μl stock in 1 ml KPE buffer) down to 0.625 μM.

NADPH solution: 2 mg NADPH;3 ml KPE buffer; Prepare fresh each time, store on ice, wrapped in aluminum foil.

Protocol for Modification of Enzyme with DTNB ^[3]:

1. Modification of the SH groups of the enzyme was 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 had been dialyzed previously against the same buffer solution for 24 h.
2. The number of SH groups was estimated from the increase of absorbance at 410 nm using a molar extinction coefficient of 13600/M×cm for thionitrobenzoate ions liberated.

This protocol only provides a guideline, and should be modified according to your specific needs.

References:

[1]. ELLMAN GL, COURTNEY KD, et,al. A new and rapid colorimetric determination of acetylcholinesterase activity. Biochem Pharmacol. 1961 Jul;7:88-95. doi: 10.1016/0006-2952(61)90145-9. PMID: 13726518.

[2]. Caito SW, Aschner M. Quantification of Glutathione in Caenorhabditis elegans. Curr Protoc Toxicol. 2015;64(618):6.18.1-6.18.6. doi: 10.1002/0471140856.tx0618s64. PMID: 26309452; PMCID: PMC4545653.

[3]. Nagaoka T, Hachimori A, Takeda A, Samejima T. DTNB modification of SH groups of isocitrate dehydrogenase from Bacillus stearothermophilus purified by affinity chromatography. J Biochem. 1977 Jan;81(1):71-8. doi: 10.1093/oxfordjournals.jbchem.a131452. PMID: 14937.