5 5 dithiobis 2 nitrobenzoicacid dtnb

Manufactured by Fujifilm

Sourced in Japan

5,5′-Dithiobis-(2-nitrobenzoicacid) (DTNB) is a chemical compound used in various laboratory applications. It is a colorimetric reagent that can be used to detect and quantify the presence of sulfhydryl groups in proteins and other biological molecules.

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6 protocols using 5 5 dithiobis 2 nitrobenzoicacid dtnb

Rice Peptide OP60: Composition and Characterization

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The commercial rice peptide
OP60 was a gift from Oryza Oil and Fat Chemical Co., Ltd. (Aichi,
Japan). OP60 is a water-soluble rice peptide produced by the enzymatic
decomposition of rice protein. The peptide content in OP60 is 68.5%,
along with 26.7% carbohydrate, 1.0% fiber, 0.1% fat, and 4.6% ash.
The tripeptide content of OP60 was estimated to be approximately 50%.
Amino acid composition, which was determined based on ion-exchange
chromatography followed by post-column derivatization with ninhydrin,
is shown Table 2.
Acetaminophen was purchased from Sigma (St. Louis,
MO, USA). 5,5′-Dithiobis-(2-nitrobenzoic
acid) (DTNB) was procured from Fujifilm Wako Pure Chemical (Osaka,
Japan). NADPH and yeast GR were purchased from Oriental Yeast Co.,
Ltd. (Tokyo, Japan). 7-Benzo-2-oxa-1,3-diazole-4-sulfonic acid (SBD-F)
and 4-(aminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole (ABD-F) were obtained
from Dojindo Labs (Kumamoto, Japan). Primary antibodies against γ-GCSh,
Nrf2, and Lamin B2 were purchased from Santa Cruz Biotechnology (Dallas,
TX, USA). Primary antibodies against HO-1 and β-actin were obtained
from Enzo Life Sciences (Farmingdale, NY) and Sigma (St. Louis, MO,
USA), respectively. The protein assay kit was obtained from Bio-Rad
Laboratories Inc. (Hercules, CA, USA).

Moritani C., Kawakami K., Shimoda H., Hatanaka T., Suzaki E, & Tsuboi S. (2020). Protective Effects of Rice Peptide Oryza Peptide-P60 against Oxidative Injury through Activation of Nrf2 Signaling Pathway In Vitro and In Vivo. ACS Omega, 5(22), 13096-13107.

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Cholinesterase Inhibitor Assay Protocol

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S-butyrylthiocholione iodide (BTC), 5-5´-dithiobis-(2-nitrobenzoic acid) (DTNB), propionylthiocholine iodide (PTC), aldicarb (98% purity), carbofuran (99.5% purity), carbaryl (99.5% purity), and propoxure (99.6%) were purchased from Wako Pure Chemical Industries (
www.wako-chem.co.jp). Phosphamidon and monocrotophos with 99.9% purity were purchased from Accustandard (
www.accustandard.com). Acetylcholine iodide (ATC), eserine, tris (hydroxymethyl) aminomethane, Triton X-100, and bovine serum albumin were obtained from Sigma-Aldrich (
www.sigmaaldrich.com).

Alizadeh A., Talebi-Jahromi K., Hosseininaveh V, & Ghadamyari M. (2014). Toxicological and biochemical characterizations of AChE in phosalone-susceptible and resistant populations of the common pistachio psyllid, Agonoscena pistaciae. Journal of Insect Science, 14, 18.

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Octacosanol and Bovine Serum Albumin Assay

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Oct (1-octacosanol), defatted bovine serum albumin, and bovine serum albumin were obtained from Sigma-Aldrich Japan Co. (Tokyo, Japan). l-Ascorbic acid, 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), 2,2'-dipyridyl, ethylenediaminetetraacetic acid (EDTA), GSH, 2-thiobarbituric acid (TBA), and other reagents of the highest grade were purchased from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan). All chemicals were used without further purification.

Ohashi K., Ohta Y., Ishikawa H, & Kitagawa A. (2020). Orally administered octacosanol improves some features of high fructose-induced metabolic syndrome in rats. Journal of Clinical Biochemistry and Nutrition, 68(1), 58-66.

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Antioxidant Assay Protocol

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CAM, H₂O₂ (30%), dimethylsulfoxide (DMSO), NaN₃, the WST-8 (2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium) assay system, and 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Mouse anti-phosphorylated ERK and anti-total ERK monoclonal antibodies, and U0126 were from Cell Signaling Technology (Tokyo, Japan). GSH, GSSG, EDTA and mouse anti-β-actin monoclonal antibody were from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). GR (from yeast) and NADPH were from Oriental Yeast Co., Ltd. (Tokyo, Japan). Dulbecco’s phosphate-buffered saline (DPBS) and phosphate-buffered saline (PBS) at pH 7.4 were from Gibco BRL (Grand Island, NY, USA). Triton-X was from IBI Scientific (Kapp Court Peosta, IA, USA). All other chemicals used were of reagent grade.

Iwayama K., Kimura J., Mishima A., Kusakabe A., Ohtaki K.I., Tampo Y, & Hayase N. (2018). Low concentrations of clarithromycin upregulate cellular antioxidant enzymes and phosphorylation of extracellular signal-regulated kinase in human small airway epithelial cells. Journal of Pharmaceutical Health Care and Sciences, 4, 23.

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Tyrosinase-Mediated Antioxidant Evaluation

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Tyrosinase (from mushrooms, specific activity 1715 U/mg), Ampliflu™ Red reagent (1-acetyl-3,7-dihydroxyphenoxazine), DA, BSA, βLG, MeCA, l-Cys and NAC were purchased from Sigma-Aldrich (St. Louis, MO, USA). FeCl₃, phenol, 3,5-di-tert-butyl-1,2-benzoquinone (DBBQ), 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB), 2-mercaptoethanol (thioglycol), reduced glutathione (GSH), thioglycolic acid, n-hexane, H₂O₂, EDTA·2Na and methanol (HPLC grade) were purchased from Wako Pure Chemical Industry (Osaka, Japan). DPRA(Cys) (Ac-RFAACAA-COOH) and DPRA(Lys) (Ac-RFAAKAA-COOH) were purchased from Scrum Inc. (Tokyo, Japan). CuSO₄·5H₂O and HClO₄ were from Katayama Chemical Co. Ltd. (Osaka, Japan). NACHis, NACLeu, NACLys and dimethyl sulfoxide (DMSO) were from Tokyo Chemical Industry Co. (Tokyo, Japan). All other chemicals were of the highest purity commercially available. The highest purity Milli-Q water (Milli-Q Advantage, Merck Millipore Co., Tokyo, Japan) was used in order to avoid contamination of metal ions. The number of thiol group in BSA and βLG were determined to be 0.30 and 0.59 mol/mol protein, respectively, using the method of Elleman [51 (link)] with DTNB. The number of thiol group in BSA was close to 0.38 mol/mol BSA [30 (link)].

Wakamatsu K., Nakao K., Tanaka H., Kitahori Y., Tanaka Y., Ojika M, & Ito S. (2019). The Oxidative Pathway to Dopamine–Protein Conjugates and Their Pro-Oxidant Activities: Implications for the Neurodegeneration of Parkinson’s Disease. International Journal of Molecular Sciences, 20(10), 2575.

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Oxidative Stress Assay Protocol

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CAM, H₂O₂ (30%), the WST-8 (2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4- disulfophenyl)-2H-tetrazolium) assay system, GSH, and 5,5’-dithiobis(2-nitrobenzoic acid) (DTNB) were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). GSH reductase (from yeast) and reduced nicotinamide adenine dinucleotide phosphate (NADPH) were from Oriental Yeast Co., Ltd. (Tokyo, Japan). Buthionine sulfoximine (BSO) and mouse anti-β-actin monoclonal antibody were from Sigma Chemical Co. (St. Louis, MO). A human IL-8 enzyme-linked immunosorbent assay (ELISA) kit was purchased from Bender Med Systems (Vienna, Austria). Rabbit polyclonal antibody against γ-GCS was from Neomarkers (Fremont, CA). The nuclear extraction and nuclear transcription factor ELISA kits were from Panomics (Fremont, CA). Cell culture media were obtained from Lonza (Walkersville, MD). All other chemicals used were of reagent grade.

Iwayama K., Kusakabe A., Ohtsu K., Nawano T., Tatsunami R., Ohtaki K.I., Tampo Y, & Hayase N. (2017). Long-term treatment of clarithromycin at a low concentration improves hydrogen peroxide-induced oxidant/antioxidant imbalance in human small airway epithelial cells by increasing Nrf2 mRNA expression. BMC Pharmacology & Toxicology, 18, 15.

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