计算溶液所需的质量、体积或浓度。
蛋白质半胱氨酸检测试剂盒 (DTNB, 微量法)
有货
为什么选择此级别
BioReagent,标曲法 级 ，适用于对基线干扰要求严格的色谱和分析工作流程。
储存与运输
2-8°C储存,避光,-20°C储存,避免反复冻融。超低温运输 。请查阅批次 COA 获取详细规格。
质量文档
SDS、COA、产品数据表及规格说明书均可下载。可通过批号查询获取批次 COA。
文献证明
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。
概述
蛋白质总巯基含量检测试剂盒 (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)以及蛋白质中的二硫键被还原后的巯基检测试剂盒。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。本试剂盒使用变性剂将样品中的蛋白进行变性，暴露二硫键和巯基，然后利用还原剂将所有二硫键进行还原，最后检测样品经变性和还原后的巯基含量，作为半胱氨酸的含量。
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，二硫键或巯基暴露，还原剂将二硫键还原成巯基(-SH)，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，可计算出样品中半胱氨酸的含量。
41、描述：
组分表
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P1527116A、P1527116B、P1527116D 可转至 2‑8 ℃存放，有效期为3个月 ②P1527116E、 P1527116F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。 |
图1. 蛋白质总巯基含量检测试剂盒 (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4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。
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图2. 蛋白质总巯基含量检测试剂盒 (DTNB, 微量法)用于GSH标准曲线的检测效果图。
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。
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！
一、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。取上清用于蛋白质半胱氨酸的测定。
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；此时测得的蛋白浓度作为CProtein-1用于后续的计算。
二、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 分析缓冲液，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。
三、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用分析缓冲液倍比稀释为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、样品处理：
(a)蛋白还原：每20μl样品中加入100μl 变性缓冲液，混匀后再加入2μl 还原缓冲液，混匀后25℃孵育1小时。
注：可根据待测样品的重复测试数量及蛋白浓度的测定需求，确定起始样品总体积。此处以一个样品20μl为例。如需测定蛋白质中二硫键的含量，本步骤需要增加未添加2μl 还原缓冲液的样品组，此时二硫键不会被还原，和添加了还原缓冲液的样品组相比，其中的差值就是由于二硫键导致的。
(b)蛋白沉淀：孵育结束后，在管中加入1ml 蛋白沉淀液，混匀后25℃孵育1小时。
(c)离心：孵育结束后，4℃ 5000×g离心15分钟，弃上清。注意不要吸取到沉淀。
(d)蛋白洗涤和重复沉淀：向沉淀中加入500μl 蛋白沉淀液，用移液器适当吹打均匀，4℃ 5000×g离心15分钟，弃上清。重复本步骤一次。注意不要吸取到沉淀。
(e)蛋白重悬：将沉淀用30ml 分析缓冲液重悬，即得到待测样品。
(f)蛋白浓度的再次测定：由于样品在沉淀处理过程中会造成蛋白损失，须取约5-10μl处理后的待测样品用BCA 蛋白浓度测定试剂盒测定蛋白浓度；此时测得的蛋白浓度作为CProtein-2用于后续的计算。
4、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)可以用分析缓冲液代替样品，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。
Blank | Standard | Sample | |
Ellmans Reagent Solution | 180μl | 180μl | 180μl |
分析缓冲液 | 20μl | － | － |
Standard | － | 20μl | － |
Sample | － | － | 20μl |
Total Volume | 200μl | 200μl | 200μl |
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。
5、 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ASample减掉ABlank，计算平均吸光度值，记录为ΔASample。
四、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蛋白质半胱氨酸浓度C。
-按样品质量计算：
C(μmol/mg)=B×D×VSample/WSample×(CProtein-1/CProtein-2)
-按样品蛋白浓度计算：
C(μmol/mg)=B×D/CProtein-2
-按半胱氨酸浓度直接计算：
C(mM)=B×D×(CProtein-1/CProtein-2)
VSample---细胞或组织裂解后的总体积，ml
WSample---样本质量，mg
CProtein-1--初始样品蛋白浓度，mg/ml
CProtein-2--处理后样品蛋白浓度，mg/ml
4、计算示例：
初始样品的蛋白浓度CProtein-1为1.6mg/ml，处理后样品蛋白浓度CProtein-2为1mg/ml，未经稀释，室温孵育15分钟。 如果测出的ASample=0.5，ABlank=0.126，则ΔASample=0.374，根据图2标准曲线公式：y=0.6895x+0.0996，计算出待测样品中的巯基浓度B=0.3980mM，则 样品中蛋白质半胱氨酸浓度C(μmol/mg)=0.3980/1=0.3980μmol/mg， 样品中半胱氨酸浓度C(mM)=0.3980×(1.6/1)=0.6368mM。
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、还原缓冲液对人体有害，操作时请小心，并注意有效防护以避免直接接触人体或吸入体内。建议在通风橱内操作。蛋白沉淀液有腐蚀性，操作时请小心，并确保有效防护以避免直接接触人体，并须注意避免腐蚀其它物品。
3、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。
4、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，3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。
Protein Total Sulfhydryl Content Assay Kit (DTNB, Micro Method) is an efficient and convenient kit for detecting exposed/surface free sulfhydryl groups, buried free sulfhydryl groups in samples including cell culture supernatants, serum, plasma, cell and tissue lysates, as well as sulfhydryl groups generated upon reduction of protein disulfide bonds. This kit employs a colorimetric (absorbance‑based) detection method.
Sulfhydryl groups (also termed thiols), chemical formula: ‑SH, are also known as hydrosulfuryl groups or mercapto groups. Sulfhydryl groups are among the highly‑reactive and widely‑existing moieties in biological systems. They are present in most proteins and also in some 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 located within the higher‑order structure of proteins. The sum of these two fractions represents total free sulfhydryl groups (unbound thiols). Ordinarily, free sulfhydryl groups in proteins refer to exposed free sulfhydryl groups, namely active sulfhydryl groups, which are predominantly harbored in cysteine residues. They play critical roles in protein structure and certain in‑vivo redox reactions. Two sulfhydryl groups in a protein can undergo dehydrogenation to form disulfide bonds (‑S‑S, Disulfide bonds), which covalently link adjacent polypeptide chains. In this kit, denaturant is used to denature proteins in samples to expose disulfide bonds and sulfhydryl groups. A reducing agent is then applied to reduce all disulfide bonds. Finally, the sulfhydryl content of denatured and reduced samples is determined, which represents cysteine content.
In biological systems, sulfhydryl groups mainly originate from glutathione and proteins. Glutathione‑derived sulfhydryl groups primarily participate in repairing oxidatively‑damaged proteins and scavenging reactive oxygen species. Protein‑derived sulfhydryl groups maintain protein conformation mainly via disulfide‑bond formation. Increased detectable free sulfhydryl groups in proteins indicate disulfide‑bond cleavage and protein structural alteration. Conventional analytical methods for sulfhydryl determination include high‑performance liquid chromatography (HPLC), capillary electrophoresis (CE), liquid chromatography‑mass spectrometry (LC‑MS), etc. Although these methods possess certain advantages in sensitivity and accuracy for free sulfhydryl detection, they suffer from relatively complicated sample handling and experimental workflows, high instrument requirements, long assay duration, and incapability of simultaneous multi‑sample measurement.
This kit adopts the classic DTNB‑based quantitative assay for free sulfhydryl groups. 5,5‑Dithio‑bis‑(2‑nitrobenzoic acid) (DTNB), also named 6,6'‑dinitro‑3,3'‑dithiobenzoic acid, Ellman Reagent, dithionitrobenzoic acid or bis(3‑carboxy‑4‑nitrophenyl) disulfide, is a water‑soluble reagent that interacts with sulfhydryl groups. It can be applied to quantify free sulfhydryl content in tissues, proteins and peptides. The fundamental reaction principle is described below: proteins in samples are subjected to denaturation, which alters native conformation, unfolds polypeptide chains and exposes disulfide bonds and sulfhydryl groups. Reducing agents convert disulfide bonds into sulfhydryl groups (‑SH). Sulfhydryl groups (R‑SH) react with DTNB to yield yellow detectable 2‑nitro‑5‑thiobenzoate anion (TNB) and mixed disulfide (R‑S‑TNB). TNB exhibits a strong absorption maximum at 412 nm. Absorbance at this wavelength is measured using a microplate reader. With glutathione as calibration standard, cysteine content in samples can be calculated.
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 P1527116A, P1527116B and P1527116D may be stored at 2‑8 °C after opening for a usable life of 3 months. ② After components P1527116E and P1527116F 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. |
Figure 1. Schematic workflow of Total Protein Sulfhydryl Content Assay Kit (DTNB, Micro‑assay).
This kit features simple sample pretreatment and fast detection. Cell culture supernatants, serum and plasma can be assayed directly. For cell and tissue samples, homogenization and lysis are performed, and supernatants are collected for measurement. The whole assay can be completed within 4 hours.
The optimized Assay Buffer of this kit chelates divalent metal ions that oxidize sulfhydryl groups, preventing sample oxidation which would lead to under‑quantification of sulfhydryl content.
This kit provides DTNB and Assay Buffer required for detection, together with GSH serving as standard or positive control.
Performance of GSH standard curve determined by this kit is shown in Figure 2. The limit of detection is approximately 10 μM.
Figure 2. GSH standard curve determined by Total Protein Sulfhydryl Content Assay Kit (DTNB, Micro‑assay).
Results may vary depending on experimental conditions and detection instruments. Data in the figure is for reference only.
For 96‑well plate format: small‑size kit supports 100‑200 tests; medium‑size kit supports 500‑1000 tests.
This product is for research use only. Not for clinical diagnosis or other applications.
Assay Procedure (For reference only)
It is recommended to run pre‑tests with 2 samples before formal experiments to characterize samples and get familiar with workflow, so as to avoid waste of samples and reagents!
I. Sample Preparation
1. Cell samples: For suspension cells, collect cells by centrifugation and wash once with PBS, HBSS or normal saline. Lyse cells using Western & IP Cell Lysis Buffer. Add 100‑200 μL lysis buffer per 1 × 10⁶ cells. For adherent cells, Western & IP Cell Lysis Buffer is also recommended; cell scraper can be used to facilitate cell collection. If recommended lysis buffer is unavailable and lysis performance is unsatisfactory, homogenize cell suspension in lysis buffer with glass homogenizer at 4 °C or on ice. Centrifuge at 12000 × g for 10 min at 4 °C. Collect supernatant for protein cysteine determination.
2. Cell culture supernatant: For adherent cells, directly collect culture supernatant for subsequent measurement. For suspension cells, collect culture supernatant after centrifugation.
3. Tissue samples: Animal tissues may be perfused with normal saline (0.9% NaCl containing 0.16 mg/mL heparin) to remove residual blood. Add 200 μL Western & IP Cell Lysis Buffer or alternative lysis buffer per 20 mg tissue. Homogenize with tissue grinder or glass homogenizer at 4 °C or on ice. Centrifuge at 12000 × g for 10 min at 4 °C, collect supernatant for subsequent assay.
4. Blood samples: For serum: incubate whole blood at room temperature (e.g. 25 °C) for 30 min‑2 h. Avoid vigorous shaking to prevent hemolysis. After natural coagulation and serum separation, centrifuge at 1000‑2000 × g for 10 min at 4 °C. Collect yellow supernatant as serum; avoid aspirating white or pale‑yellow precipitate. For plasma: anticoagulate whole blood with heparin or EDTA. Centrifuge at 1000‑2000 × g for 10 min at 4 °C. Collect yellow or pale‑yellow supernatant as plasma; avoid aspirating white precipitate.
5. Determine protein concentration of above samples using BCA Protein Concentration Assay Kit. The measured protein concentration is denoted as C<sub>Protein‑1</sub> for subsequent calculation.
II. Kit Reagent Preparation
1. Preparation of 8 mM GSH Standard: Dissolve 4 mg GSH in 1.627 mL Assay Buffer and mix thoroughly to obtain 8 mM GSH Standard. Dispense GSH Standard into aliquots and store at −20 °C.
2. Preparation of DTNB Stock Solution: Dissolve 2 mg DTNB in 0.5 mL Assay Buffer and mix thoroughly to prepare DTNB Stock Solution. Dispense DTNB Stock Solution into aliquots and store at −20 °C.
3. Preparation of Ellman’s Reagent Solution: 180 μL Ellman’s Reagent Solution is required per sample. Calculate total volume according to sample number. Mix DTNB Stock Solution with Denaturation Buffer at volume ratio 1:35 to prepare Ellman’s Reagent Solution. Example: mix 50 μL DTNB Stock Solution with 1.75 mL Denaturation Buffer to yield ~1.8 mL Ellman’s Reagent Solution.
Note: Prepare Ellman’s Reagent Solution freshly according to actual consumption.
III. Sample Measurement
1. Warm‑up microplate reader for ≥30 min and set wavelength to 412 nm.
2. Standard curve setup: Serially dilute 8 mM GSH Standard with Assay Buffer to 0, 0.125, 0.25, 0.5, 1, 2, 4 mM. Refer to Figure 2 for GSH standard curve. Appropriate GSH concentrations can also be self‑defined for standard curve construction.
3. Sample processing
(a) Protein reduction: Add 100 μL Denaturation Buffer to 20 μL sample, mix well, add 2 μL Reduction Buffer, mix and incubate at 25 °C for 1 h.
Note: Starting sample volume can be adjusted according to replicate number and protein concentration requirement. Here 20 μL sample is taken as example. To quantify disulfide‑bond content in protein, set an additional sample group without adding 2 μL Reduction Buffer, where disulfide bonds remain unreduced. The difference value between reduced group and non‑reduced group corresponds to disulfide‑bond‑derived sulfhydryl content.
(b) Protein precipitation: After incubation, add 1 mL Protein Precipitation Solution to each tube, mix and incubate at 25 °C for 1 h.
(c) Centrifugation: Upon incubation completion, centrifuge at 5000 × g for 15 min at 4 °C. Discard supernatant. Do not aspirate pellet.
(d) Protein washing & repeated precipitation: Add 500 μL Protein Precipitation Solution to pellet, pipette up‑and‑down for thorough resuspension. Centrifuge at 5000 × g for 15 min at 4 °C and discard supernatant. Repeat this step once. Do not aspirate pellet.
(e) Protein resuspension: Resuspend pellet with 30 mL Assay Buffer to obtain treated test sample.
(f) Re‑determination of protein concentration: Protein loss occurs during precipitation. Take 5‑10 μL treated sample and measure protein concentration with BCA Protein Concentration Assay Kit. This value is denoted as C<sub>Protein‑2</sub> for subsequent calculation.
4. Add 180 μL Ellman’s Reagent Solution into each well. Pipette 20 μL standard or treated sample into 96‑well plate as shown in table below. Assay Buffer can substitute sample for Blank Control; alternatively absorbance of zero‑concentration standard point can serve as blank. Incubate at room temperature (20‑25 °C) for 15 min.
| Item | Blank Control | Standard | Sample |
|---|---|---|---|
| Ellman’s Reagent Solution | 180 μL | 180 μL | 180 μL |
| Assay Buffer | 20 μL | — | — |
| Standard | — | 20 μL | — |
| Sample | — | — | 20 μL |
| Total Volume | 200 μL | 200 μL | 200 μL |
Note 1: For reliable results, 2‑3 replicate wells are recommended for each sample.
Note 2: If sulfhydryl content exceeds linear range of standard curve, dilute sample appropriately with Assay Buffer or lysis buffer and record dilution factor D. If sulfhydryl content is too low, increase input amount of tissue or cell material during sample preparation.
Note 3: The above system is 200 μL. If sulfhydryl concentration is sufficiently high, reagent volumes can be halved to double total test number.
5. Measure absorbance at 412 nm by microplate reader. Record absorbance of blank control as ABlank; absorbance of samples or standards as ASample or AStandard. Subtract ABlank from ASample and calculate mean absorbance denoted as ΔASample.
IV. Result Calculation
1. Calculate mean absorbance AStandard for each standard point. Subtract ABlank to obtain net absorbance for each standard. Plot standard curve with GSH concentration (mM) as abscissa and absorbance as ordinate.
2. Substitute ΔASample into standard‑curve formula to calculate sulfhydryl concentration (denoted as B).
3. Select appropriate formula according to experimental requirement to calculate protein‑bound cysteine concentration C.
- Calculation based on sample mass
C(μmol/mg) = B × D × VSample / WSample × (CProtein‑1 / CProtein‑2)
- Calculation based on sample protein concentration
C(μmol/mg) = B × D / CProtein‑2
- Direct cysteine concentration calculation
C(mM) = B × D × (CProtein‑1 / CProtein‑2)
VSample: total volume of cell/tissue lysate, mL
WSample: sample weight, mg
CProtein‑1: initial protein concentration of sample, mg/mL
CProtein‑2: protein concentration of treated sample, mg/mL
4. Calculation example Initial protein concentration CProtein‑1 = 1.6 mg/mL; post‑treatment protein concentration CProtein‑2 = 1 mg/mL; no dilution; 15 min room‑temperature incubation. If measured ASample = 0.5, ABlank = 0.126, ΔASample = 0.374. According to standard‑curve equation y = 0.6895x + 0.0996, calculated sulfhydryl concentration B = 0.3980 mM. Protein‑bound cysteine concentration: C(μmol/mg) = 0.3980 / 1 = 0.3980 μmol/mg Sample cysteine concentration: C(mM) = 0.3980 × (1.6 / 1) = 0.6368 mM
Precautions
1. This assay is redox‑dependent. Any oxidizing or reducing agents will interfere with measurement. If reducing agents in samples (e.g. DTT, β‑mercaptoethanol) cannot be avoided, keep their total concentration lower than intrinsic sulfhydryl concentration of samples. Common detergents such as Triton X‑100 and Tween‑20 contain high levels of peroxides. If detergents are mandatory, select high‑purity grades specified with low peroxide content.
2. Reduction Buffer is hazardous to human health. Handle with caution and wear adequate personal protective equipment to avoid skin contact and inhalation. Manipulation inside fume hood is recommended. Protein Precipitation Solution is corrosive. Handle carefully with proper protection to prevent bodily contact and damage to labware.
3. This product is intended for scientific research performed by qualified personnel only. Not for clinical diagnosis, therapy, food or pharmaceutical applications. Do not store in residential premises.
4. Wear lab coat and disposable gloves for personal safety and health.
Shelf‑life
Ship under cold conditions. Stable for 2 years stored at −20 °C; stable for 3 months stored at 4 °C.
After GSH and DTNB are prepared into solutions, dispense appropriately and store at −20 °C; the prepared solutions are stable for at least 3 months.
规格
规格或纯度
BioReagent, 标曲法
英文名称
Protein Cysteine Assay Kit (DTNB, Micro Method)
应用
细胞代谢
检测方法
分光光度法
检测仪器
酶标仪
检测波长
412nm
敏感性
Light Sensitive
储存与运输
储存条件
2-8°C储存,避光,-20°C储存,避免反复冻融
运输条件
超低温运输
稳定性与储存
Store at -20℃ long term (24 months).
组分与储存
| 组分货号 | 组分名称 | 物理外观 | 100T | 500T | 储存 |
| P1527116A | 分析缓冲液 | 液体 | 50ml | 250ml | 2-8℃ |
| P1527116B | 变性缓冲液 | 液体 | 20ml | 100ml | 2-8℃ |
| P1527116C | 还原缓冲液 | 液体 | 200μl | 1ml | -20℃ |
| P1527116D | 蛋白沉淀液 | 液体 | 200ml | 500ml×2 | 2-8℃ 避光 |
| P1527116E | GSH | 固体 | 4mg | 20mg | -20℃ |
| P1527116F | DTNB | 固体 | 2mg | 10mg | 2-8℃ |
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P1527116A、P1527116B、P1527116D 可转至 2‑8 ℃存放，有效期为3个月
②P1527116E、 P1527116F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。
技术文档
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技术文档和文章
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