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DTNB (Ellman's Reagent)

Catalog No.GC30009

DTNB (Ellman's Reagent) (Ellman's Reagent) is a chemical used to quantify the number or concentration of thiol groups.

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DTNB (Ellman's Reagent) Chemical Structure

Cas No.: 69-78-3

Size Price Stock Qty
10mM (in 1mL DMSO)
$46.00
In stock
1g
$41.00
In stock
5g
$60.00
In stock

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Sample solution is provided at 25 µL, 10mM.

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Product Documents

Quality Control & SDS

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Protocol

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.

Background

DTNB (Ellman's reagent) is an abbreviation for 5,5'-Dithiobis(2-nitrobenzoic acid). When it reacts with thiol compounds, colorless DTNB changes into yellow 5-thio-2-nitrobenzoic acid. This transformation is useful because 5-thio-2-nitrobenzoic acid has its maximum absorption at 412 nm, ensuring that DTNB's absorption spectrum doesn't interfere with thiol determination. Consequently, DTNB is commonly employed to measure thiol content in proteins and peptide tissues. It can also be used for monitoring organophosphorus pesticide poisoning through acetylcholinesterase assays[1-3].

DTNB modifies the SH group of the enzyme, enabling the determination of the number of SH groups in each molecule and their contribution to the activity of the enzyme[4]. Using a hydroxycinnamoyl-CoA:l-DOPA hydroxycinnamoyl transferase as a model, DTNB has little to no effect on the transferase reaction and can be used to provide a good estimate of hydroxycinnamoyl amide formation, thus allowing for the quick and easy collection of reaction rate data and determination of transferase kinetic parameters[5]. DTNB modification facilitated crystallization of CcbP by inducing polar interactions in the crystal lattice. DTNB-mediated cysteine modification was demonstrated to have little effect on the overall structure and the Ca2+ binding of CcbP. DTNB modification may provide a simple and general approach for protein modification to improve the success of crystallization screening[6].

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]. ELLMAN GL. Tissue sulfhydryl groups. Arch Biochem Biophys. 1959 May;82(1):70-7. doi: 10.1016/0003-9861(59)90090-6. PMID: 13650640.

[3]. Tietze F. Enzymic method for quantitative determination of nanogram amounts of total and oxidized glutathione: applications to mammalian blood and other tissues. Anal Biochem. 1969 Mar;27(3):502-22. doi: 10.1016/0003-2697(69)90064-5. PMID: 4388022.

[4]. Nagaoka T, Hachimori A, 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. doi: 10.1093/oxfordjournals.jbchem.a131452. PMID: 14937.

[5]. Sullivan ML, Bonawitz ND. Spectrophotometric determination of reaction rates and kinetic parameters of a BAHD acyltransferase using DTNB (5,5'-dithio-bis-[2-nitrobenzoic acid]). Plant Sci. 2018 Apr;269:148-152. doi: 10.1016/j.plantsci.2018.01.012. Epub 2018 Jan 31. PMID: 29606213.

[6]. Fan XX, Zhou YF, et,al. Ellman's reagent in promoting crystallization and structure determination of Anabaena CcbP. Acta Crystallogr Sect F Struct Biol Cryst Commun. 2012 Nov 1;68(Pt 11):1409-14. doi: 10.1107/S1744309112034938. Epub 2012 Oct 30. PMID: 23143261; PMCID: PMC3515393.

Chemical Properties

Cas No. 69-78-3 SDF
Canonical SMILES O=C(O)C1=C([N+]([O-])=O)C=CC(SSC2=CC(C(O)=O)=C([N+]([O-])=O)C=C2)=C1
Formula C14H8N2O8S2 M.Wt 396.35
Solubility DMSO : ≥ 43 mg/mL (108.49 mM) Storage Store at -20°C
General tips Please select the appropriate solvent to prepare the stock solution according to the solubility of the product in different solvents; once the solution is prepared, please store it in separate packages to avoid product failure caused by repeated freezing and thawing.Storage method and period of the stock solution: When stored at -80°C, please use it within 6 months; when stored at -20°C, please use it within 1 month.
To increase solubility, heat the tube to 37°C and then oscillate in an ultrasonic bath for some time.
Shipping Condition Evaluation sample solution: shipped with blue ice. All other sizes available: with RT, or with Blue Ice upon request.

Complete Stock Solution Preparation Table

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1 mg 5 mg 10 mg
1 mM 2.523 mL 12.6151 mL 25.2302 mL
5 mM 0.5046 mL 2.523 mL 5.046 mL
10 mM 0.2523 mL 1.2615 mL 2.523 mL
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Method for preparing DMSO master liquid: mg drug pre-dissolved in μL DMSO ( Master liquid concentration mg/mL, Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug. )

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