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DTNB (Ellman's Reagent) Sale

(Synonyms: Ellman试剂; Ellman’s Reagent) 目录号 : GC30009

DTNB (Ellman’s Reagent) 是一种用于定量巯基数量或浓度的化学物质。常用于测量蛋白质和肽组织中的硫醇含量(SH含量)。

DTNB (Ellman's Reagent) Chemical Structure

Cas No.:69-78-3

规格 价格 库存 购买数量
10mM (in 1mL DMSO)
¥446.00
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1g
¥401.00
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5g
¥580.00
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Sample solution is provided at 25 µL, 10mM.

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实验参考方法

DTNB使用说明 [1]:

标准溶液(10mm):避光处理,首先称出0.198克DTNB,在50 mM Na2HPO4 (pH 7.0)中配制50 mL溶液。将溶液保存在棕色或琥珀色的瓶子中,并保存在阴凉(4℃),黑暗的地方备用。

秀丽隐杆线虫总谷胱甘肽的测定 [2]:

  1. 在96孔板上每孔加20 μl KPE空白缓冲液、20 μl GSH标准品或20 μl样品。空白、标准品和样品一式两份。
  2. 将DTNB和GR溶液按1:1比例混合。用多通道移液器向每孔中加入120 μl。
  3. 等待30秒后,用多通道移液器向每孔中加入60 μl NADPH溶液。
  4. 立即在412 nm处每30秒读取吸光度2分钟。
  5. 计算标准品和样品的2-硝基-5-硫代苯甲酸的生成速率(即每分钟吸光度的变化)。
  6. 绘制标准浓度与2-硝基-5 -硫代苯甲酸生成速率的关系曲线。
  7. 采用标准曲线线性回归法测定样品井中谷胱甘肽浓度。
  8. 使用提取物的剩余部分定量谷胱甘肽提取物的蛋白质含量。

DTNB solution: 2 mg DTNB;3 ml KPE buffer; 现用现配,放冰上,用铝箔包裹(避光)。

KPE buffer (pH 7.5):  6.8 g KH2PO4加500 ml无菌水,4℃保存;溶液B: 11.4 g K2HPO4-3H2O加500 ml无菌水,4℃保存;4℃保存,可保存1个月。

Glutathione reductase (GR) solution: 40 μl GR (250 U/ml);3 ml KPE buffer; 现用现配,放冰上。

GSH standards: Stock 2 mM GSH: :6.14 mg谷胱甘肽于10 ml KPE缓冲液中,1ml分装,在-20℃下保存1个月。从40 μM GSH (20 μl原液在1 ml KPE缓冲液中)开始连续稀释至0.625 μM。

NADPH solution: 2 mg NADPH;3 ml KPE buffer; 现用现配,放冰上,用铝箔包裹(避光)。

DTNB修饰酶[3]:

  1. 将10 μl 10 mM DTNB溶液(约20倍摩尔过量)与0.6 ml酶溶液(0.807 mg/ml)在80 mM磷酸盐缓冲液(pH 8.0,含1 mM EDTA)中反应24 h,在室温下对酶的SH基团进行修饰。
  2. 利用释放的亚硝基苯甲酸酯离子的摩尔消光系数13600/M×cm,用410 nm处吸光度的增加估计出SH基团的数量。

仅供参考,请根据您的实验具体需要进行修改。

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.

产品描述

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.

DTNB (Ellman's reagent)是5,5'-二硫比斯(2-硝基苯甲酸)的缩写。当它与硫醇类化合物反应时,无色的DTNB变成黄色的5-硫-2-硝基苯甲酸。5-硫代-2-硝基苯甲酸在412 nm处有最大吸收,DTNB的吸收光谱不会干扰硫醇的测定。因此,DTNB通常用于测量蛋白质和肽组织中的硫醇含量。通过乙酰胆碱酯酶测定,也可用于有机磷农药中毒的监测[1-3]

DTNB可以修饰酶的SH基团,能够确定每个分子中SH基团的数量及其对酶活性的贡献[4]。以羟肉桂酰辅酶a:l-DOPA羟肉桂酰转移酶为模型,DTNB对转移酶反应几乎没有影响,可以很好地估计羟肉桂酰酰胺的形成,从而可以快速方便地收集反应速率数据和确定转移酶动力学参数[5]。DTNB改性通过诱导晶格中的极性相互作用促进了CcbP的结晶。DTNB介导的半胱氨酸修饰被证明对CcbP的整体结构和Ca2+结合几乎没有影响。DTNB修饰可为蛋白质修饰提供一种简单而通用的方法,从而提高结晶筛选的成功率[6]

Chemical Properties

Cas No. 69-78-3 SDF
别名 Ellman试剂; Ellman’s Reagent
Canonical SMILES O=C(O)C1=C([N+]([O-])=O)C=CC(SSC2=CC(C(O)=O)=C([N+]([O-])=O)C=C2)=C1
分子式 C14H8N2O8S2 分子量 396.35
溶解度 DMSO : ≥ 43 mg/mL (108.49 mM) 储存条件 Store at -20°C
General tips 请根据产品在不同溶剂中的溶解度选择合适的溶剂配制储备液;一旦配成溶液,请分装保存,避免反复冻融造成的产品失效。
储备液的保存方式和期限:-80°C 储存时,请在 6 个月内使用,-20°C 储存时,请在 1 个月内使用。
为了提高溶解度,请将管子加热至37℃,然后在超声波浴中震荡一段时间。
Shipping Condition 评估样品解决方案:配备蓝冰进行发货。所有其他可用尺寸:配备RT,或根据请求配备蓝冰。

溶解性数据

制备储备液
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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*在配置溶液时,请务必参考产品标签上、MSDS / COA(可在Glpbio的产品页面获得)批次特异的分子量使用本工具。

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动物体内配方计算器 (澄清溶液)

第一步:请输入基本实验信息(考虑到实验过程中的损耗,建议多配一只动物的药量)
给药剂量 mg/kg 动物平均体重 g 每只动物给药体积 ul 动物数量
第二步:请输入动物体内配方组成(配方适用于不溶于水的药物;不同批次药物配方比例不同,请联系GLPBIO为您提供正确的澄清溶液配方)
% DMSO % % Tween 80 % saline
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Research Update

Ellman's Reagent prevents dephosphorylation of histones during isolation of mitotic chromosomes

Chromosome Res 2022 Dec;30(4):351-359.36399199 10.1007/s10577-022-09709-1

Histones H1 and H3 are highly phosphorylated in mitotic HeLa cells but are rapidly dephosphorylated by endogenous protein phosphatases during the isolation of metaphase chromosomes. We show that this dephosphorylation can be prevented by including the sulfhydryl reagent 5,5'-dithiobis-(2-nitrobenzoate) (Ellman's Reagent, or DTNB) in the isolation buffer. The minimal amount of DTNB required is approximately stoichiometric with the number of sulfhydryl groups in the lysate. Inhibition of the protein phosphatases can subsequently be reversed by treatment with dithiothreitol or 2-mercaptoethanol. DTNB is compatible with the isolation of either metaphase chromosome clusters or individual metaphase chromosomes. It should be useful in investigations of the structure and biochemistry of chromatin and chromosomes and in the study of possible functions for mitotic histone phosphorylation.

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):1409-14.23143261 PMC3515393

Obtaining crystals presented a bottleneck in the structural study of Anabaena cyanobacterial Ca2+-binding protein (CcbP). In this report, the promoting effect of Ellman's Reagent [5,5'-dithiobis(2-nitrobenzoic acid); DTNB] on the crystallization of CcbP is described. CcbP contains one free cysteine. A quick and simple oxidation reaction with DTNB blocked the free cysteine in purified CcbP and generated a homogenous monomeric protein for crystallization. The crystal structure of DTNB-modified CcbP was determined by the single-wavelength anomalous diffraction method. Structure analysis indicated that 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. Thus, DTNB modification may provide a simple and general approach for protein modification to improve the success of crystallization screening.

Reassessment of Ellman's Reagent

Methods Enzymol 1983;91:49-60.6855597 10.1016/s0076-6879(83)91010-8

Ellman's Reagent: 5,5'-dithiobis(2-nitrobenzoic acid)--a reexamination

Anal Biochem 1979 Apr 1;94(1):75-81.37780 10.1016/0003-2697(79)90792-9

Thiol-disulfide exchange reactions occurring at modified bovine serum albumin detected using Ellman's Reagent (5, 5'-dithiobis (2-itrobenzoic acid)

Pak J Pharm Sci 2020 Nov;33(6(Supplementary)):2767-2772.33879435

Bovine serum albumin (BSA) is usually employed as a model protein because of being homologous with human serum albumin. Cysteine-34 of BSA has been oxidised with Ellman's Reagent to produce BSA labelled with an Ellman's moiety (BSA-SE). The BSA-SE was then reacted with glutathione, N-acetylcysteine and D-penicillamine (D-pen). The two were able to release the Ellman's moiety bound at cysteine-34 while D-pen did not. Albumin labeled using Ellman's Reagent was used to demonstrate the cleavage of a protein mixed disulphide. The kinetics of thiol disulfide interchange reactions involving formation of a chromophoric thiolate were determined by UV-visible spectroscopy. The reaction of thiolates with excess Ellman's Reagent is used for quantitative estimation of thiol by measuring the absorption at 位, 412 nm. The disulfide exchange reactions occurring at Cys-34 of BSA was determined and the reduction of oxidized Cys-34 was studied in order to understand the reverse reaction. Spectroscopic evidence suggested that glutathione and N-acetylcysteine remove the label and produce BSA in a disulfide form. In contrast, D-pen reaction returned BSA to its thiolate form via mediation. It was observed that thio-disulfide exchange occurred at cysteine-34 labelled with Ellman's moiety. The implications to the redox status of plasma are discussed.