BioReagent 级 ，适用于对基线干扰要求严格的色谱和分析工作流程。
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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。
生物体内巯基主要包括谷胱甘肽巯基和蛋白质巯基。前者不仅能够修复氧化损伤的蛋白质，而且参与活性氧清除，后者对于维持蛋白质构象具有重要作用。通过测定总巯基含量和GSH含量，能够间接测定蛋白质巯基含量。
检测原理：巯基基团与5,5’-二硫代-双-硝基苯甲酸（DTNB）反应，生成黄色化合物，在412 nm处有最大吸收峰。
注：本试剂盒仅限科研使用，不可用于临床诊断及其他用途。
标准品（T1515982D）注意事项
自备仪器和试剂
操作步骤（仅供参考）
1. 样本提取
1.1 组织样本
称取约0.1 g组织，加入1 mL提取液，进行冰浴匀浆。然后10000 rpm，4℃离心10 min，取上清置 冰上待测。
注：
① 根据实验室条件，可先液氮研磨，再加提取液，进行冰浴匀浆。
② 根据研究需求，可按组织质量（g）：提取液体积（mL）为1:10的比例进行提取。
1.2 液体样本
澄清的液体样本直接检测，若浑浊则离心后再取上清液检测。
2. 检测步骤
2.1 可见分光光度计预热30 min（等仪器过自检程序亦可），设定波长为412 nm，蒸馏水调零。
2.2 所有试剂在使用前均须在室温或25℃水浴锅中温育10 min。
2.3 在EP管或1 mL玻璃比色皿（光径1 cm）中依次加入：
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注：
① 若加入试剂二有白色浑浊产生，立即混匀样本即可恢复澄清。
② 若A测定值大于1.5可减少样本加样体积V1（如由30 μL减至10 μL），试剂一相应增加，或者把上清液用蒸馏水稀释后测定，则改变的V1和D需带入公式计算。
3. 结果计算
3.1 标准曲线方程：y=3.4274x+0.0008，x为标准品摩尔浓度 (μmoL/mL)；y为ΔA。
3.2 样本总巯基含量计算
① 按样本质量计算
总巯基含量 (μmoL/g鲜重)=[(ΔA-0.0008)÷3.4274×V1]÷(W×V1÷V)×D=0.2918×(ΔA-0.0008)÷W×D
② 按蛋白浓度计算
总巯基含量 (μmoL/mg prot)=[(ΔA-0.0008)÷3.4274×V1]÷(Cpr×V1÷V)×D =0.2918×(ΔA-0.0008)÷Cpr×D
③ 按液体体积计算
总巯基含量 (μmoL/mL)=[(ΔA-0.0008)÷3.4274×V1]÷V1×D =0.2918×(ΔA-0.0008)×D
④ 按细菌/细胞数量计算
总巯基含量 (μmoL/10 <sup>4</sup> cell)=[(ΔA-0.0008)÷3.4274×V1]÷(500×V1÷V)×D=0.2918×(ΔA-0.0008)÷500×D
参数说明：
V：加入提取液体积，1 mL
V1：加入样本体积，0.12 mL
W：样本质量，g
GSH分子量：307.3
D：稀释倍数，未稀释即为1
500：细菌或细胞总数，万
Cpr：上清液蛋白浓度，mg/mL，推荐使用阿拉丁B665595 BCA蛋白定量试剂盒或R1491648 即用型BCA蛋白定量试剂盒。
标准曲线制作过程
1. 标曲为非必做实验，用户可根据实验需求制作标曲，亦可直接采用说明书计算公式进行结果计算。
2. 制备标准品母液（1 μmol/mL）：标准品中加入2 mL蒸馏水，充分溶解，即1 μmol/mL标准品母液。
3. 将母液用蒸馏水稀释成六个浓度梯度的标准品，例如：0，0.1，0.2，0.3，0.4，0.5 μmoL/mL。也可根据实际样本调整标准品浓度。
4. 标品稀释参照表如下：
吸取标准品母液500 μL，加入500 μL蒸馏水，混匀得到0.5 μmoL/mL的标品稀释液待用。
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5. 依据测定管的加样表操作，根据结果，过0点制作标准曲线。
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Sulfhydryl groups in organisms mainly include glutathione sulfhydryl (GSH) and protein sulfhydryl groups. The former not only repairs oxidatively damaged proteins but also participates in the scavenging of reactive oxygen species, while the latter plays an important role in maintaining protein conformation. By measuring total sulfhydryl content and GSH content, protein sulfhydryl content can be indirectly determined.
Detection Principle:
Sulfhydryl groups react with 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) to form a yellow compound with a maximum absorption peak at 412 nm.
Note: For research use only. Not for clinical diagnosis or other purposes.
Standard (T1515982D) Precautions:
Use this standard if you need to prepare a new standard curve.
Prepare according to the standard curve preparation instructions.
Dissolved standard should be used within one week.
Reagents, consumables and Equipments not provided
Protocol (for reference only):
1. Sample Preparation
1.1 Tissue samples:
Weigh approximately 0.1 g of tissue, add 1 mL of Extraction Buffer, and homogenize on ice. Centrifuge at 10,000 rpm for 10 min at 4°C. Collect the supernatant and keep on ice until use.
Notes:
① If available, grind tissue under liquid nitrogen before adding Extraction Buffer and homogenizing on ice.
② According to research needs, extraction can be performed at a ratio of tissue mass (g) to Extraction Buffer volume (mL) = 1:10.
1.2 Liquid samples:
Clear liquid samples can be directly measured. If turbid, centrifuge and use the supernatant for measurement.
2. Assay Procedure
2.1 Preheat the visible spectrophotometer for 30 min (or allow the instrument to complete its self-check), set the wavelength to 412 nm, and zero with distilled water.
2.2 All reagents should be warmed to room temperature or in a 25°C water bath for 10 min before use.
2.3 Add the following reagents to an EP tube or a 1 mL glass cuvette (1 cm light path) in order:
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Notes:
① If a white turbidity appears after adding Reagent 2, mix the sample immediately to restore clarity.
② If AAssay > 1.5, reduce the sample volume V1 (e.g., from 30 μL to 10 μL) and increase Reagent 1 accordingly, or dilute the supernatant with distilled water before measurement. The changed V1 and dilution factor (D) must be included in the calculation.
3. Calculation
3.1 Standard curve equation: y = 3.4274x + 0.0008, where x = standard concentration (μmol/mL), y = ΔA.
3.2 Calculation of total sulfhydryl content in samples:
(1) Based on sample mass (fresh weight):
Total sulfhydryl content (μmol/g fresh weight) = [(ΔA – 0.0008) ÷ 3.4274 × V1] ÷ (W × V1 ÷ V) × D
= 0.2918 × (ΔA – 0.0008) ÷ W × D
(2) Based on protein concentration:
Total sulfhydryl content (μmol/mg prot) = [(ΔA – 0.0008) ÷ 3.4274 × V1] ÷ (Cpr × V1 ÷ V) × D
= 0.2918 × (ΔA – 0.0008) ÷ Cpr × D
(3) Based on liquid sample volume:
Total sulfhydryl content (μmol/mL) = [(ΔA – 0.0008) ÷ 3.4274 × V1] ÷ V1 × D
= 0.2918 × (ΔA – 0.0008) × D
(4) Based on bacterial/cell count:
Total sulfhydryl content (μmol/10<sup>4</sup> cells) = [(ΔA – 0.0008) ÷ 3.4274 × V1] ÷ (500 × V1 ÷ V) × D
= 0.2918 × (ΔA – 0.0008) ÷ 500 × D
Parameters:
V = volume of Extraction Buffer added = 1 mL
V1 = sample volume added = 0.12 mL
W = sample mass (g)
Molecular weight of GSH = 307.3
D = dilution factor (if undiluted, D = 1)
500 = total number of bacteria or cells (×10<sup>4</sup>)
Cpr = protein concentration in the supernatant (mg/mL). It is recommended to use Aladdin B665595 BCA Protein Assay Kit or R1491648 Ready-to-Use BCA Protein Assay Kit.
Standard Curve Preparation
1. Preparing a standard curve is not mandatory. Users may either generate a standard curve or directly use the formula provided in the manual for calculation.
2. Prepare standard stock solution (1 μmol/mL): Add 2 mL of distilled water to the standard vial and dissolve completely. This yields a 1 μmol/mL standard stock solution.
3. Dilute the stock solution with distilled water to six concentration gradients, e.g., 0, 0.1, 0.2, 0.3, 0.4, 0.5 μmol/mL. Concentrations may be adjusted according to actual samples.
4. Standard dilution reference table:
Pipette 500 μL of standard stock solution, add 500 μL distilled water, mix to obtain 0.5 μmol/mL diluted standard.
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5. Operate according to the Assay Tube addition table, and generate the standard curve passing through zero.
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| T1515982 | Component | 100T/48S | Storage |
| T1515982A | 提取液 | 60 mL | 2-8℃. |
| T1515982B | 试剂一 | 55 mL | 2-8℃. |
| T1515982C | 试剂二 | 4 mL | 2-8℃. Store in the dark. |
| T1515982D | 标准品 | 1 EA | 2-8℃. |
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| 批号(Lot Number) | 证书类型 | 货号 |
|---|---|---|
 ZJ26F0737384 |
分析证书 | T1515982 |
| ZJ26F0434889 |
分析证书 | T1515982 |
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