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。
谷胱甘肽是一种含γ-酰胺键和巯基的三肽，由谷氨酸、半胱氨酸及甘氨酸组成，广泛存在于动、植物组织和微生物中，在生物体内能帮助免疫系统保持正常的功能，并具有抗氧化和整合解毒的作用。谷胱甘肽有还原型（GSH）和氧化型（GSSG）两种形式，GSH 是细胞内最主要的抗氧化巯基物质，在抗氧化、蛋白质巯基保护和氨基酸跨膜运输等过程中具有重要作用，还原型与氧化型谷胱甘肽含量的比值（GSH/GSSG）可作为评估细胞氧化还原状态的主要动态指标。
检测原理：还原型谷胱甘肽能够与5,5’-二硫代-双-（2-硝基苯甲酸）（5,5’-Dithiobis-2-Nitrobenoic acid，DTNB） 反应生成黄色的2-硝基-5-巯基苯甲酸（TNB）和谷胱甘肽二硫化物（GSSG），TNB在 412 nm 处具有特征吸收峰，通过吸光值变化即可定量检测还原型谷胱甘肽的含量。
自备仪器和试剂
实验流程
建议先选取1-3个差异大的样本（例如不同类型或分组）进行预实验，熟悉操作流程，根据预实验 结果确定或调整样本浓度，以防造成样本或试剂不必要的浪费！
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。4℃×12000 rpm 离心15 min，取上清液，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 细菌/细胞样本
先收集细菌或细胞到离心管内，离心后弃上清；取500万细菌或细胞加入1 mL提取液；超声波破碎细菌或细胞（功率300 w，超声3 s，间隔7 s，总时间3 min）；4℃×12000 rpm 离心15 min，取上清液，置冰上待测。
注：若增加样本量，可按照细菌/细胞数量（10⁴个）：提取液（mL）为500~1000：1比例进行提取。
1.3 液体样本
直接检测。若浑浊，离心后取上清检测。
2. 检测步骤
2.1 可见分光光度计预热30 min，调节波长到412 nm，蒸馏水调零。
2.2 所有试剂在使用前需在25℃水浴中保温10 min。
2.3 在1 mL玻璃比色皿（光径1 cm）中依次加入：
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注：
(1) 若是同一批样本检测，样本对照管做一个即可。
(2) 若ΔA值在零附近徘徊，可以增加样本取样质量（如增至0.2 g）或加大上样量（如增至160 μL，试剂一相应减少）。
3. 标准曲线制作
3.1 标准品溶于2 mL蒸馏水中（母液需在两天内用且-20℃保存），标准品母液浓度为1 μmol/mL。将母液用蒸馏水稀释成六个浓度梯度的标准品，例如：0、0.2、0.4、0.6、0.8、1 μmol/mL。也可根据实际样本调整标准品浓度。
3.2 标品稀释参照表如下：
| 标准品工作液 | 标准品母液（μL） | 水（μL） | 浓度（µmol/mL） |
| Std.1 | 0 | 200 | 0 |
| Std.2 | 40 | 160 | 0.2 |
| Std.3 | 80 | 120 | 0.4 |
| Std.4 | 120 | 80 | 0.6 |
| Std.5 | 160 | 40 | 0.8 |
| Std.6 | 200 | 0 | 1.0 |
注：各标准管混匀待用。
3.3 依据测定管的加样表操作，根据结果，以各浓度吸光值减去0浓度吸光值，制作标准曲线。
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4. 结果计算
4.1 标准曲线为y=1.5674x-0.015，R²=0.9993；x为标准品浓度（μmol/mL），y为ΔA。
4.2 样本GSH含量计算
(1) 按蛋白浓度计算
GSH (μmol/mg prot)=[(ΔA+0.015)÷1.5674×V1]÷(V1×Cpr)=0.638×(ΔA+0.015)÷Cpr
(2) 按样本鲜重计算
GSH (μmol/g鲜重)=[(ΔA+0.015)÷1.5674×V1]÷(W×V1÷V)=0.638×(ΔA+0.015)÷W
(3) 按细胞数量计算
GSH (μmol/ 10⁴ cell)=[(ΔA+0.015)÷1.5674×V1]÷(500×V1÷V)=0.638×(ΔA+0.015)÷500
(4) 按液体体积计算
GSH (μmol/mL)=[(ΔA+0.015)÷1.5674×V1]÷V1=0.638×(ΔA+0.015)
参数说明:
V：上清液总体积，1 mL；
V1：加入反应体系中上清液体积，80 μL=0.08 mL；
W：样品质量，g；
500：细菌/细胞数量，万；
GSH分子量：307.3；
Cpr：蛋白质浓度，mg/mL。
注意事项
1. 上清液不能用于蛋白质浓度测定。如需测定蛋白含量，需另取相同样本。如需测定蛋白浓度，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。
2. 生化检测试剂普遍具有刺激性、生物毒性等，为了您的安全和健康，请在实验全程做好生物安全防护，穿戴实验服、口罩、手套、头套等安全护具，在通风橱、生物安全柜中进行实验。
3. 本产品仅供科学研究使用，不适用于临床诊断。
Glutathione is a tripeptide containing a γ‑amide bond and a sulfhydryl group, composed of glutamate, cysteine, and glycine. It is widely distributed in animal and plant tissues as well as microorganisms. In living organisms, it helps maintain normal immune function and possesses antioxidant and detoxifying effects. Glutathione exists in two forms: reduced (GSH) and oxidized (GSSG). GSH is the major antioxidant thiol compound within cells and plays important roles in antioxidation, protection of protein thiol groups, and transmembrane amino acid transport. The ratio of reduced to oxidized glutathione (GSH/GSSG) serves as a key dynamic indicator for assessing cellular redox status.
Detection Principle: Reduced glutathione (GSH) reacts with 5,5'‑dithio‑bis‑(2‑nitrobenzoic acid) (DTNB) to produce yellow 5‑thio‑2‑nitrobenzoic acid (TNB) and glutathione disulfide (GSSG). TNB exhibits a characteristic absorption peak at 412 nm. The GSH content can be quantified by measuring the change in absorbance.
Reagents, consumables and Equipments not provided
Experimental Workflow
It is recommended to first perform a pilot experiment using 1–3 samples with large differences (e.g., different types or groups) to familiarize yourself with the procedure and determine or adjust sample concentrations based on the pilot results, to avoid unnecessary waste of samples or reagents.
1. Sample Extraction
1.1 Tissue Samples
Weigh approximately 0.1 g of tissue, add 1 mL of Extraction Solution, and homogenize on ice. Centrifuge at 4°C, 12,000 rpm for 15 minutes. Collect the supernatant and keep on ice for assay.
Notes:
(1) Depending on laboratory conditions, samples may first be ground in liquid nitrogen before adding Extraction Solution and homogenizing on ice.
(2) According to research needs, extraction can be performed at a tissue mass (g) to Extraction Solution volume (mL) ratio of 1:10.
1.2 Bacterial/Cell Samples
Collect bacteria or cells into a centrifuge tube, centrifuge, and discard the supernatant. Take 5 million bacteria or cells and add 1 mL of Extraction Solution. Sonicate to disrupt the bacteria/cells (power 300 W, pulse 3 s, interval 7 s, total time 3 minutes). Centrifuge at 4°C, 12,000 rpm for 15 minutes. Collect the supernatant and keep on ice for assay.
Note: If increasing sample amount, extract at a ratio of bacteria/cell count (10⁴ cells) to Extraction Solution (mL) of 500–1000:1.
1.3 Liquid Samples
Assay directly. If turbid, centrifuge and use the supernatant.
2. Assay Procedure
2.1 Preheat the visible spectrophotometer for 30 minutes, set wavelength to 412 nm, and zero with distilled water.
2.2 Warm all reagents in a 25°C water bath for 10 minutes before use.
2.3 In a 1 mL glass cuvette (1 cm path length), add in order:
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Notes:
(1) For the same batch of samples, one sample control tube is sufficient.
(2) If ΔA is near zero, increase the sample amount (e.g., to 0.2 g tissue) or increase the sample volume added (e.g., to 160 μL, with Reagent 1 reduced accordingly).
2. Standard Curve Preparation
3.1 Dissolve the Standard in 2 mL of distilled water (the stock solution should be used within two days and stored at –20°C). The stock concentration is 1 μmol/mL. Dilute the stock with distilled water to create six concentration gradients, e.g., 0, 0.2, 0.4, 0.6, 0.8, 1.0 μmol/mL. Adjust standard concentrations according to actual sample levels.
3.2 Standard dilution reference table:
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3.3 Follow the addition table for the test tubes. Using the results, subtract the absorbance of the 0‑concentration tube from each standard absorbance and construct a standard curve.
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4. Result Calculation
4.1 Standard curve: y = 1.5674x – 0.015, R² = 0.9993; where x is standard concentration (μmol/mL), y is ΔA.
4.2 Sample GSH Content Calculation
(1) Based on Protein Concentration
GSH (μmol/mg prot) = [(ΔA + 0.015) ÷ 1.5674 × V₁] ÷ (V₁ × Cpr) = 0.638 × (ΔA + 0.015) ÷ Cpr
(2) Based on sample fresh weight:
GSH (μmol/g fresh weight) = [(ΔA + 0.015) ÷ 1.5674 × V₁] ÷ (W × V₁ ÷ V) = 0.638 × (ΔA + 0.015) ÷ W
(3) Based on cell count:
GSH (μmol/10⁴ cells) = [(ΔA + 0.015) ÷ 1.5674 × V₁] ÷ (500 × V₁ ÷ V) = 0.638 × (ΔA + 0.015) ÷ 500
(4) Based on Liquid Volume
GSH (μmol/mL) = [(ΔA + 0.015) ÷ 1.5674 × V₁] ÷ V₁ = 0.638 × (ΔA + 0.015)
Parameter Explanation:
V: Total volume of supernatant (1 mL)
V₁: Volume of supernatant added to the reaction system (80 μL = 0.08 mL)
W: Sample mass (g)
500: Bacteria/cell count, in 10⁴ units
GSH Molecular Weight: 307.3
Cpr: Protein concentration (mg/mL)
Precautions
The supernatant cannot be used for protein concentration determination. If protein content needs to be measured, take a separate aliquot of the same sample. For protein concentration determination, Aladdin B665595 BCA Protein Quantification Kit or R1491648 Ready‑to‑Use BCA Protein Quantification Kit is recommended.
Biochemical assay reagents are generally irritating and biologically toxic. For your safety and health, maintain appropriate biosafety precautions throughout the experiment: wear lab coats, masks, gloves, head covers, etc., and perform experiments in a fume hood or biosafety cabinet.
This product is for scientific research only and not suitable for clinical diagnosis.
| R1505409 | Component | 48T | Storage |
| R1505409A | 提取液 | 50 mL | 2-8℃. Store in the dark. |
| R1505409B | 试剂一 | 60 mL | 2-8℃. |
| R1505409C | 试剂二 | 8 mL | 2-8℃. Store in the dark. |
| R1505409D | 标准品 | 1 EA | 2-8℃. |
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| 批号(Lot Number) | 证书类型 | 货号 |
|---|---|---|
 ZJ26F0636123 |
分析证书 | R1505409 |
| ZJ26F0232356 |
分析证书 | R1505409 |
危险品化学品经营许可证（带存储）