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在色谱分析、有机合成和交叉偶联反应领域已被 0 篇同行评审文献引用。
总游离巯基检测试剂盒 (DTNB, 微量法) ，是一种高效、便捷的用于细胞培养上清液、血清、血浆、细胞及组织裂解液样品中总游离巯基含量的检测试剂盒，即暴露的或表面的游离巯基(Exposed/Surface free sulfhvdryl groups)和埋藏于蛋白内部的游离巯基(Buried free sulfhydryl groups)的检测试剂盒。本试剂盒采用显色法(吸光度法)进行检测。
巯基(Sulfhydryl groupsor Thiols)，又称氢硫基或硫醇基，化学式为-SH。巯基是生物系统中反应性非常强并且广泛存在的基团之一，它存在于大多数蛋白质中，也存在于一些低分子量物质中，如谷胱甘肽(GSH)、COA、脂酸、DTT、巯基乙酸盐和游离半胱氨酸等。蛋白质中的游离巯基通常分为暴露的游离巯基(Exposed/Surface free sulfhydryl groups)和在蛋白质高级结构中的游离巯基(Buried free sulthydryl groups)，两者之和为总游离巯基(Total free sulthydryl groups or unbound thiols)。通常蛋白质中的游离巯基指的是暴露的游离巯基，即活性巯基，主要存在于半胱氨酸中，是蛋白质结构和生物体内某些氧化还原反应的重要基团，在蛋白质中两个巯基脱氢而形成的二硫键(-S-S,Disulfide bonds)，使相邻多肽得以连接。本试剂盒可以同时检测暴露的游离巯基和在蛋白质高级结构中的游离巯基，即总游离巯基的检测，但不会检测已经形成二硫键的巯基。
生物体内的巯基主要为谷胱甘肽巯基和蛋白质巯基，其中谷胱甘肽巯基主要参与修复氧化损伤的蛋白质和活性氧的清除；蛋白质巯基则主要通过形成二硫键发挥维持蛋白质构象的作用，而蛋白质中检测到增多的游离巯基则提示二硫键发生断裂，蛋白质结构发生变化。目前，巯基传统的测定方法主要有高效液相色谱法(HPLC)、毛细管电泳法(CE)、液相色谱-质谱联用法(HPLG-GC)等。虽然这些方法在游离巯基检测的灵敏度和准确度上有一定优势，但是样品处理和实验操作相对复杂、对仪器的要求较高、检测耗时较长、且无法同时检测多个样品。
本试剂盒采用经典的基于DTNB的游离巯基定量检测方法。5,5-二硫代双(2-硝基苯甲酸)(5,5-Dithio-bis-(2-nitrobenzoic acid)，DTNB)，又称为6,6'-二硝基-3,3'-二硫代苯甲酸、Ellman试剂(Ellman Reagent)、二硫代硝基苯甲酸或双(3-羧基-4-硝基苯基)二硫，是一种与巯基相互作用的水溶性试剂，可用于组织、蛋白质和多肽中游离巯基含量的测定，基本反应原理如下：样品中的蛋白质通过变性处理，原有构像发生改变，蛋白质肽链松散开，从而使原来埋藏于蛋白质内部的巯基(-SH)暴露，所有游离巯基(R-SH)与DTNB反应生成黄色可检测的2-硝基-5-硫代苯甲酸盐阴离子(TNB)和混合二硫化物(R-S-TNB)，TNB在412nm处有强吸收峰，通过酶标仪测定此处的吸光度，并以谷胱甘肽为标准品，可计算出样品中总游离巯基含量。
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图1. 游离巯基检测试剂盒 (DTNB, 微量法)工作原理图。
本试剂盒样品处理简单，检测速度快。对于细胞培养上清液、血清、血浆可直接检测；对于细胞及组织样品只需匀浆裂解，取上清进行检测。整个检测过程不足半小时即可完成。
本试剂盒的分析缓冲液经过优化，可螯合可以氧化巯基的二价金属离子，防止样品被氧化导致样品中巯基含量低于实际值。
本试剂盒提供了检测所需的DTNB和 分析缓冲液 ，同时还提供GSH作为标准品或阳性对照(Positive control)。
本试剂盒检测GSH标准曲线的效果如图2所示。最低检测限约为10μM。
图2. 游离巯基检测试剂盒 (DTNB, 微量法)用于GSH标准曲线的检测效果图。
实测数据会因实验条件、检测仪器等的不同而存在差异，图中数据仅供参考。
用于96孔板检测时，本试剂盒小包装的可以进行100-200次检测，中包装可以进行500-1000次检测。
本产品仅用于科研领域，不适用于临床诊断或其他用途。
组分表
① 整套试剂盒未拆封可于−20 ℃避光保存，自生产之日起有效期 24 个月。为避免反复冻融，开封后组分 F1527507A、 F1527507B可转至 2‑8 ℃存放，有效期为6个月 ② F1527507C、 F1527507D配制成溶液后，需适当分装，-20℃保存至少3个月有效； ③固体量在 mg 级别，使用前用手甩几次或者进行离心，打开直接加入要求的试剂即可。 |
操作步骤(仅供参考)
建议正式实验前，选取 2 个样本做预测定，了解实验样品情况，熟悉流程，避免样本和试剂浪费。
样品的准备：
1、细胞样品的准备：对于悬浮细胞，可以离心收集细胞，并用PBS、HBSS或生理盐水洗涤一遍。后续可以用Western及IP细胞裂解液裂解并制备细胞样品。按照每100万细胞直接加入100-200微升裂解液的比例进行裂解。对于贴壁细胞，也推荐使用Western及IP细胞裂解液进行裂解，同时可以使用细胞铲或细胞刮辅助收集细胞样品。如果没有使用推荐的裂解液，出现裂解效果不佳的情况，可以把处在裂解液中的细胞样品用玻璃匀浆器在4℃或冰浴匀浆。4℃ 12000×g离心10分钟。取上清用于总巯基的测定。
2、细胞培养液上清的准备：对于贴壁细胞，直接吸取培养液上清用于后续的测定；对于悬浮细胞，离心后吸取培养液上清用于后续的测定。
3、组织样品的准备：动物组织可以酌情使用含有0.16mg/ml 肝素的生理盐水(0.9% NaCl containing 0.16mg/ml heparin)灌流清除组织上残留的血液。每20mg组织加入200μl Western及IP细胞裂解液或其它的裂解液，用组织研磨仪或玻璃匀浆器在4℃或冰浴等条件下匀浆。4℃ 12000×g离心10分钟，取上清用于后续的测定。
4、血液样品的准备：对于血清样品，将全血在常温下(如25℃)放置30分钟至2小时，不要剧烈摇晃以免溶血，待全血自然凝固并析出血清后，4℃ 1000-2000×g离心10分钟，取黄色上清即得血清，注意不要吸取白色或淡黄色沉淀；对于血浆样品，将全血用肝素或者EDTA进行抗凝，4°C 1000-2000×g离心10分钟，取黄色或淡黄色上清即得血浆，注意不要吸取白色沉淀。
5、上述样品可以用BCA蛋白浓度测定试剂盒测定蛋白浓度。
试剂盒的准备工作：
1、GSH Standard (8mM)的配制：每4mg GSH中加入1.627ml 分析缓冲液，溶解并混匀，即为8mM的GSH Standard。GSH Standard适当分装后于-20°C保存。
2、DTNB储存液的配制：每2mg DTNB中加入0.5ml Assay Buffer，溶解并混匀，即为DTNB储存液。DTNB储存液适当分装后于-20°C保存。
3、Ellman's Reagent Solution的配制：每个样品需180μl Ellman's Reagent Solution，请根据样品数量计算所需Ellman's Reagent Solution的总量。将DTNB储存液与变性缓冲液按照1:35的比例混匀，即得Ellman's Reagent Solution。例如取50μI DTNB储存液加入1.75ml 变性缓冲液，混匀即得约1.8ml Ellman's Reagent Solution。
注：Ellman's Reagent Solution需根据用量配制，现用现配。
样品测定：
1、酶标仪预热30min以上，调节波长至 412nm。
2、标准曲线的设置：将8mM的GSH Standard用Assay Buffer倍比稀释为0、0.125、0.25、0.5、1、2、4mM。GSH标准曲线请参考图2。也可自行设置适宜的GSH浓度进行标准曲线的设定。
3、每孔加入180μI Ellman's Reagent Solution，参考下表分别将20μI的标准品或者待测样品加入到96孔板中。空白对照(Blank Control)可以用f缓冲液代替样品，也可以不设置而使用标准曲线中浓度为0的吸光度值。室温(约20-25°C)孵育15分钟。
注 1：为获取更加可靠的检测结果，推荐每个样品设置 2‑3 个平行孔。
注 2：如果样品中巯基含量过高，超出标准曲线检测范围，可用分析缓冲液或裂解液对样品进行适当稀释，稀释倍数记录为 D。如果样品中巯基含量过低，则在样品制备时使用更多的组织或细胞样品。
注 3：上述推荐的测定体系为 200μl，如果样品中的巯基浓度不是太低，完全可以把每种试剂的用量减半，这样试剂盒可以测定的总次数可以加倍。
4、用酶标仪测定 412nm 处的吸光度，测得的空白对照吸光度值，记录为 ABlank，测得的样品或标准品吸光度值，记录为 ASample或 AStandard，用 AStandard减掉 ABlank，计算平均吸光度值，记录为 ΔASample。
结果计算
1、计算标准品组中每个点的平均吸光度 AStandard，用 AStandard减掉 ABlank，即为各个标准品的吸光度。以 GSH 标准 (mM) 为横坐标，吸光度为纵坐标，绘制出标准曲线。
2、将 ΔASample代入标准曲线，即可计算出巯基浓度 (记录为 B)。
3、根据实验需求选择适当的公式计算总巯基浓度 C。 按样品质量计算： C (μmol/mg)=B×D×VSample /WSample 按样品蛋白浓度计算： C (μmol/mg)=B×D /CProtein 按巯基浓度直接计算： C (mM)=B×D VSample---- 细胞或组织裂解后的总体积，ml WSample---- 样本质量，mg CProtein--- 样品蛋白浓度，mg/ml
4、计算示例：样品的蛋白浓度 CProtein经测定为 1mg/ml，未经稀释，室温孵育 15 分钟。如果测出的 ASample=0.312，ABlank=0.064，则 ΔASample=0.248，根据图 2 标准曲线公式：y=0.6294x+0.1002，计算出待测样品中的总巯基浓度 B=0.256mM，则待测样品中总巯基浓度 C (μmol/mg)=0.256/1=0.256umol/mg。
注意事项
1、本试剂盒检测时涉及氧化还原反应，因此所有氧化剂或还原剂都会干扰本试剂盒的测定。如果在样品中的还原剂 (例如 DTT、巯基乙醇等) 无法避免，则应尽量控制还原剂的总浓度低于待测样品中的巯基浓度。常用的 Triton X‑100、Tween‑20 等去垢剂都含有较高水平的过氧化物，如果必须使用去垢剂，最好使用纯度较高并注明含较低过氧化物的去垢剂。
2、本产品仅限于专业人员的科学研究用，不得用于临床诊断或治疗，不得用于食品或药品，不得存放于普通住宅内。
3、为了您的安全和健康，请穿实验服并戴一次性手套操作。
有效期
低温运输。‑20℃保存，两年有效；4℃保存，6 个月有效。
GSH 和 DTNB 配制成溶液后，需适当分装，‑20℃保存至少 3 个月有效。
Total Free Sulfhydryl Assay Kit (DTNB, Micro Method), Also designated as Total Sulfhydryl Assay Kit with DTNB or Total Thiol Assay Kit with DTNB, this kit is also known as Total Sulfhydryl Content Assay Kit or Total Sulfhydryl Quantification Assay Kit. It is an efficient and convenient kit for determining total free sulfhydryl groups in cell culture supernatants, serum, plasma, cell lysates and tissue lysates, including exposed/surface free sulfhydryl groups and buried free sulfhydryl groups within proteins. This kit adopts colorimetric (absorbance‑based) detection.
Sulfhydryl groups (also termed thiols), chemical formula: ‑SH. As highly reactive and ubiquitous moieties in biological systems, sulfhydryl groups exist in most proteins as well as low‑molecular‑weight substances such as glutathione (GSH), CoA, fatty acids, DTT, thioglycolate and free cysteine. Free sulfhydryl groups in proteins are generally divided into exposed/surface free sulfhydryl groups and buried free sulfhydryl groups within the higher‑order protein structure. Their sum is defined as total free sulfhydryl groups (unbound thiols). Ordinarily, free sulfhydryl groups in proteins refer to exposed free sulfhydryl groups (active sulfhydryl groups), predominantly located in cysteine residues. They are critical for protein structure and multiple biological redox reactions. Two sulfhydryl groups in proteins can undergo dehydrogenation to form disulfide bonds (‑S‑S‑), which covalently link adjacent polypeptide chains. This kit simultaneously detects both exposed and buried free sulfhydryl groups within higher‑order protein structures (i.e., total free sulfhydryl groups), yet it does not measure sulfhydryl groups that have already formed disulfide bonds.
Biological sulfhydryl groups mainly consist of glutathione‑derived thiols and protein‑derived thiols. Glutathione thiols primarily participate in repairing oxidatively damaged proteins and scavenging reactive oxygen species. Protein thiols maintain protein conformation mainly via disulfide‑bond formation. Increased detectable free sulfhydryl groups in proteins indicate disulfide‑bond cleavage and protein structural perturbation. Conventional analytical approaches for sulfhydryl determination include high‑performance liquid chromatography (HPLC), capillary electrophoresis (CE), and liquid chromatography‑mass spectrometry (LC‑MS). Although these methods deliver satisfactory sensitivity and accuracy for free sulfhydryl measurement, they suffer from labor‑intensive sample pretreatment, high instrument requirements, long assay duration, and low throughput for parallel sample testing.
This kit employs the classic DTNB‑based quantitative method for free sulfhydryl determination. 5,5‑Dithio‑bis‑(2‑nitrobenzoic acid) (DTNB), also named Ellman’s Reagent, is a water‑soluble thiol‑reactive reagent for quantifying free sulfhydryl groups in tissues, proteins and peptides. Assay principle: proteins in samples are subjected to denaturation to disrupt native conformation and unfold polypeptide chains, thereby exposing buried internal ‑SH moieties. All free thiols (R‑SH) react with DTNB to yield yellow 2‑nitro‑5‑thiobenzoate anion (TNB) and mixed disulfide (R‑S‑TNB). TNB exhibits a strong absorption maximum at 412 nm. Absorbance is read with a microplate reader, and total free sulfhydryl concentration is calculated against a glutathione standard curve.
Figure 1. Schematic workflow of Free Sulfhydryl Assay Kit (DTNB, microplate method).
This kit features simple sample handling and rapid detection. Cell culture supernatants, serum and plasma can be assayed directly. Cell and tissue samples only require homogenization‑lysis, followed by supernatant collection for measurement. The whole procedure can be finished within half an hour.
The optimized Assay Buffer chelates divalent metal ions that oxidize thiols, preventing artifactual thiol loss and under‑quantification.
DTNB and Assay Buffer are supplied together with GSH serving as standard and positive control.
Performance of the GSH standard curve is shown in Figure 2. Limit of detection ≈10 μM.
Figure 2. Representative GSH standard curve obtained with Free Sulfhydryl Assay Kit (DTNB, microplate method).
Actual readings may vary depending on experimental conditions and instruments; the figure is for reference only.
For 96‑well microplate format: small‑size kit supports 100‑200 tests; large‑size kit supports 500‑1000 tests.
For research use only. Not for clinical diagnosis or other clinical applications.
Component
Notes: ① The complete unopened kit shall be stored at −20 °C protected from light, with a shelf‑life of 24 months from the date of manufacture. To avoid repeated freeze‑thaw cycles, components F1527507A, F1527507B may be stored at 2‑8 °C after opening for a usable life of 6 months. ② After components F1527507C and F1527507D are prepared into solutions, aliquot appropriately and store at −20 °C; they remain valid for at least 3 months. ③ The solid contents are at the mg level. Vial‑tap several times by hand or centrifuge before use, then open the vial and directly add the required reagents. |
Assay Procedure (For reference only)
It is recommended to run a preliminary test with 2 samples prior to formal experiments to evaluate samples and familiarize with workflow and avoid reagent/sample waste.
Sample Preparation
1. Cell samples: Collect suspension cells by centrifugation and wash once with PBS, HBSS or normal saline. Lyse cells using Western & IP lysis buffer (100‑200 μL lysis buffer per 1 × 10⁶ cells). For adherent cells, use Western & IP lysis buffer and harvest cells with cell scraper. If lysis is insufficient, homogenize cell suspension with glass homogenizer on ice / at 4 ℃. Centrifuge at 12 000 × g for 10 min at 4 ℃, retain supernatant for total sulfhydryl assay.
2. Cell culture supernatant. Directly harvest medium supernatant for adherent cells; centrifuge suspension‑cell culture and collect supernatant for measurement.
3. Tissue samples: Animal tissues may be perfused with 0.9% NaCl containing 0.16 mg/mL heparin to remove residual blood. Add 200 μL Western & IP lysis buffer (or alternative lysis buffer) per 20 mg tissue, homogenize with tissue homogenizer or glass homogenizer on ice / at 4 ℃. Centrifuge at 12 000 × g for 10 min at 4 ℃, collect supernatant.
4. Blood samples: For serum: allow whole blood to stand 30 min‑2 h at room temperature (e.g. 25 ℃). Avoid vigorous mixing to prevent hemolysis. After clot formation, centrifuge at 1 000‑2 000 × g for 10 min at 4 ℃. Collect yellow supernatant (serum); avoid white/pale‑yellow pellet. For plasma: anticoagulate whole blood with heparin or EDTA. Centrifuge at 1 000‑2 000 × g for 10 min at 4 ℃, collect pale‑yellow plasma supernatant; avoid white pellet.
5. Determine sample protein concentration using BCA protein assay kit.
Reagent Preparation
1. Preparation of 8 mM GSH Standard: Dissolve 4 mg GSH powder in 1.627 mL Assay Buffer and mix well to obtain 8 mM GSH Standard. Aliquot and store at ‑20 ℃.
2. Preparation of DTNB Stock Solution: Dissolve 2 mg DTNB in 0.5 mL Assay Buffer and mix well. Aliquot DTNB stock and store at ‑20 ℃.
3. Preparation of Ellman’s Reagent Solution: 180 μL Ellman’s Reagent Solution is required per well. Calculate total volume according to sample number. Mix DTNB stock solution with Denaturing Buffer at 1:35 (v/v). Example: mix 50 μL DTNB stock with 1.75 mL Denaturing Buffer to yield ~1.8 mL working solution.
Note: Prepare Ellman’s Reagent Solution freshly before use according to required volume.
Sample Measurement
1. Pre‑warm microplate reader ≥30 min, set wavelength to 412 nm.
2. Standard curve setup: Serially dilute 8 mM GSH Standard with Assay Buffer to obtain 0, 0.125, 0.25, 0.5, 1, 2, 4 mM. Refer to Figure 2; alternative concentration series are acceptable.
3. Add 180 μL Ellman’s Reagent Solution to each well. Dispense 20 μL standard or sample into 96‑well plate as shown below. For blank control, use Assay Buffer instead of sample; alternatively use zero‑concentration point of standard curve. Incubate 15 min at room temperature (20‑25 ℃).
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Note
1: For reliable results, run 2‑3 technical replicates per sample.
2: If sulfhydryl concentration exceeds linear range, dilute sample with Assay Buffer or lysis buffer; record dilution factor as D. If sulfhydryl content is low, increase input amount of tissue/cells during sample preparation.
3: Recommended total reaction volume is 200 μL. If sample thiol concentration is sufficiently high, all reagent volumes can be halved to double total assay throughput.
4. Read absorbance at 412 nm with microplate reader. Record blank absorbance as ABlank; sample / standard absorbance as ASample / AStandard. Calculate ΔASample = ASample − ABlank.
Calculation
1. Compute average absorbance AStandard for each standard point; subtract ABlank to obtain net absorbance. Plot standard curve with GSH concentration (mM) on x‑axis and net absorbance on y‑axis.
2. Substitute ΔASample into standard‑curve equation to calculate sample sulfhydryl concentration, denoted as B.
3. Select appropriate formula for total sulfhydryl concentration C: Calculation by sample mass: C (μmol/mg) = B × D × VSample / WSample Calculation by sample protein concentration: C (μmol/mg) = B × D / CProtein Direct concentration output: C (mM) = B × D VSample: total volume of cell/tissue lysate, mL WSample: sample weight, mg CProtein: sample protein concentration, mg/mL
4. Calculation example: Sample protein concentration CProtein = 1 mg/mL, no dilution, 15 min room‑temperature incubation. ASample = 0.312, ABlank = 0.064, ΔASample = 0.248. Standard‑curve equation: y = 0.6294x+ 0.1002. Calculated B = 0.256 mM. C (μmol/mg) = 0.256 / 1 = 0.256 μmol/mg.
1. This assay is redox‑sensitive. Oxidizing and reducing agents interfere with detection. If reducing agents such as DTT or β‑mercaptoethanol cannot be avoided in samples, keep their total concentration below endogenous thiol concentration. Detergents including Triton X‑100 and Tween‑20 often contain high peroxide levels. If detergents are required, select high‑purity grades with low peroxide content.
2. For research use by trained personnel only. Not for clinical diagnosis, therapeutic use or food‑pharmaceutical application. Do not store in residential premises.
3. Wear lab coat and disposable gloves for personal safety.
Shelf‑life & Storage
Ship under cold conditions. Stable for 2 years stored at −20 ℃; stable for 6 months stored at 4 ℃.
After reconstitution, aliquot GSH and DTNB solutions; stable ≥3 months at −20 ℃.
注： ① 整套试剂盒未拆封可于−20 ℃避光保存，自生产之日起有效期 24 个月。为避免反复冻融，开封后组分 F1527507A、 F1527507B可转至 2‑8 ℃存放，有效期为6个月。 ② F1527507C、 F1527507D配制成溶液后，需适当分装，-20℃保存至少3个月有效。 ③固体量在 mg 级别，使用前用手甩几次或者进行离心，打开直接加入要求的试剂即可。 |
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