谷胱甘肽(glutathione)是一种由3个氨基酸残基组成的小肽，全称为谷氨酰-半胱氨酰-甘氨酸，英文名称为glutamyl-cysteinyl glycine，简称为glutathione。谷胱甘肽包括还原型谷胱甘肽 (Reduced Glutathione, GSH)和氧化型谷胱甘肽 (Oxidized Glutathione, GSSG)。其中氧化型谷胱甘肽 (GSSG)是还原型谷胱甘肽 (GSH)的氧化形式，又称为二硫代谷胱甘肽，是两分子的谷胱甘肽氧化而成。GSSG会被谷胱甘肽还原酶还原成GSH，因此机体中大多数是以还原型形式存在。测定细胞内GSH和GSSG含量以及GSH/GSSG比值，能够很好地反映细胞所处的氧化还原状态。
本试剂盒检测原理是利用谷胱甘肽还原酶 (Glutathione Reductase，GR)将GSSG还原为GSH，GSH与5,5-二硫双 (2-硝基苯甲酸）(DTNB)反应生成2-硝基-5-巯基苯甲酸，反应液呈黄色，测定其在412 nm处的吸光度值的变化即可计算样本中总谷胱甘肽的含量。然后通过2-乙烯吡啶抑制样品中原有的还原型谷胱甘肽，然后利用上述反应即可测定样本中 GSSG 的含量，从总谷胱甘肽中减去氧化型谷胱甘肽的含量即可计算出样本中还原型谷胱甘肽的含量。
G1373339 | 组件 | 物料外观 | 100T | 储存 | 单次测试使用量 |
G1373339A | 检测缓冲液 | 无色液体 | 14 mL | -20℃. | 133 µL |
G1373339B | 提取液 | 无色液体 | 60 mL | -20℃. | 按需 |
G1373339C | 抑制剂 | 淡黄色液体 | 200 µL | -20℃.避光. | 2 µL |
G1373339D | 谷胱甘肽还原酶 | 无色液体 | 200 µL | -20℃.避光. | 2 µL |
G1373339E | 辅因子 | 粉末 | 8.4 mg | -20℃.避光. | 按需 |
G1373339F | 显色液 | 淡黄色液体 | 4 mL | -20℃.避光. | 40 µL |
G1373339G | GSSG标准品 | 无色液体 | 1 mL | -20℃.避光. | 按需 |
注意：抑制剂有毒，建议在通风橱内操作。
使用方法
1. 自备试剂耗材
(1) 96 孔板，1.5 mL离心管，匀浆器或者超声破碎仪。
(2) 37℃恒温培养箱或者水浴锅，酶标仪。
(3) 无水乙醇，无菌去离子水，PBS。
2. 试剂准备
(1) 辅因子工作液配制：将8.4 mg辅因子粉末用1 mL无菌去离子水完全溶解，终浓度10 mM。在使用时建议分装成多支小管，-20℃避光保存，避免反复冻融。
(2) 抑制剂工作液配制：取1体积抑制剂+2体积无水乙醇混匀后即可，-20℃避光保存。
(3) 谷胱甘肽还原酶在冰上解冻，检测缓冲液，提取液和显色液可平衡至室温。
(4) 标准品配制：取10 μL GSSG标准品（1.0 mM）+ 90 μL提取液稀释至100 μM，然后再用提取液分别稀释至40 μM，20 μM，10 μM，5 μM，2.5 μM，1.25 μM，0.625 μM和0 μM。建议每次实验都重新制作标准曲线。
3. 待测总谷胱甘肽含量样品的制备
(1) 组织样本：请尽量使用新鲜的组织样本进行测定。每10 mg组织样本加入100-200 μL提取液，快速冰上匀浆（匀浆器提前放冰上预冷）。匀浆后的样本，4℃，8,000 g 离心10 min，取20 μL上清液，置冰上待测。
(2) 血液样品：对于血清样品，将全血在常温下静置30分钟-2小时，4ºC约1000-2000×g离心10分钟，取上清；对于血浆样品，将全血用肝素或者EDTA进行抗凝，4ºC约1000-2000×g离心10分钟，取20 μL上清待测。
(3) 细胞（菌）样本：请尽量使用新鲜的细胞（菌）进行测定，而不要使用冻存的细胞（菌）进行测定。收集个细胞（菌），首先用预冷的PBS 清洗细胞（菌）2 次（PBS 重悬，4℃ 500 g 离心 5 min）， 1×10⁶ 个细胞（菌）建议加入100-200 μL提取液重悬，冰浴超声波破碎 (200W，超声 2s，间隔 3s，5min)，4℃，8,000 g 离心 5 min，取20 μL上清液，置冰上待测。
4. 待测GSSG含量样品的制备
(1) 取部分前面准备好的待测总谷胱甘肽含量的样品至1.5 mL EP管中，按照每28 μL样品加入2 μL抑制剂工作液，混匀后，37℃避光孵育15 min，离心，取20 μL上清置冰上待测。
注意：添加抑制剂时，需在1.5 mL EP管内进行添加，不能直接在96孔板中添加，抑制剂会直接腐蚀96孔板，影响消除效率。
5. 样品和标准品的测定
(1) 在96孔板中，依照下表依次加入样品或者标准品
试剂 | 空白孔 (μL) | 测定孔 (μL) | 标准孔 (μL) |
样本 | - | 20 | - |
去离子水 | 20 | - | - |
标准品 | - | - | 20 |
检测缓冲液 | 133 | 133 | 133 |
谷胱甘肽还原酶 | 2 | 2 | 2 |
辅因子工作液 | 5 | 5 | 5 |
显色液 | 40 | 40 | 40 |
(2) 混匀后，于37℃避光孵育10 min，然后读取 A₄₁₂ 。
Δ标准=A测定 - A空白；Δ标准=A标准 - A空白
6. 结果计算
(1) 标准曲线：y=0.0617*X -0.0134(仅供参考)；x是GSSG的浓度(μM)，y 是△A。
(2) 可根据标准曲线分别计算出样品中的总谷胱甘肽浓度 (C) 和GSSG浓度 (C)，计算公示如下：
C (μM)=A×n
注：A为根据标准曲线计算出的GSSG浓度 (μM)；n为样品稀释倍数。
(3) 根据测定得到的总谷胱甘肽的含量和GSSG的含量就可以计算出GSH的含量。计算公式为:GSH=(总谷胱甘肽含量-GSSG含量)×2。因为1个GSSG分子在反应后可以还原成2个GSH分子。比如根据标准曲线分别测得总谷胱甘肽和GSSG的浓度分别为7.5 μM和1.2 μM，那么GSH浓度应该为 (7.5-1.2)×2=12.6 μM。
注意事项
1. 辅因子配制完后记得分装-20℃避光保存，抑制剂，GSSG标准品和显色液同样避光保存。
2. 抑制剂有毒，建议在通风橱内操作。
3. 添加抑制剂消除GSH时，需在1.5 mLEP内进行添加，不能直接在96孔板中添加，抑制剂会直接腐蚀96孔板，影响消除效率。
4. 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。
Glutathione is a small peptide composed of three amino acid residues, formally named γ-glutamyl-cysteinyl-glycine. Its abbreviated common name is glutathione. Glutathione exists in two forms: reduced glutathione (GSH) and oxidized glutathione (GSSG). Oxidized glutathione (GSSG), also known as glutathione disulfide, is the oxidized dimer formed by two molecules of reduced glutathione linked via a disulfide bond. GSSG can be converted back to GSH catalyzed by glutathione reductase, so GSH is the predominant form of glutathione in organisms. Quantification of intracellular GSH, GSSG and the GSH/GSSG ratio serves as a reliable biomarker for evaluating cellular redox status.
This kit employs glutathione reductase (GR) to reduce GSSG into GSH. The generated GSH reacts with 5,5'-dithio-bis-(2-nitrobenzoic acid) (DTNB) to produce 2-nitro-5-thiobenzoate, a yellow chromogenic product. The total glutathione concentration in samples is calculated by measuring the absorbance change at 412 nm. To separately quantify GSSG content, endogenous GSH in samples is masked by pretreatment with 2-vinylpyridine. The aforementioned chromogenic reaction is then performed to determine GSSG levels. The concentration of reduced glutathione (GSH) is obtained by subtracting the quantified GSSG concentration from the total glutathione concentration.
G1373338 | Components | Appearance | 100T | Storage | Usage volume per test |
G1373338A | Assay Buffer | Colorless liquid | 14 mL | -20℃. | 133 µL |
G1373338B | Extraction Buffer | Colorless liquid | 60 mL | -20℃. | As need |
G1373338C | Inhibitor | light yellow liquid | 200 µL | -20℃.Store in the dark. | 2 µL |
G1373338D | GR | Colorless liquid | 200 µL | -20℃.Store in the dark. | 2 µL |
G1373338E | Cofactor | Powder | 8.4 mg | -20℃.Store in the dark. | As need |
G1373338F | Chromogen | light yellow liquid | 4 mL | -20℃.Store in the dark. | 40 µL |
Note: The inhibitor is toxic. All operations should be carried out in a fume hood.
Usage Method
1. Self-supplied Reagents and Consumables
(1) 96-well plate, 1.5 mL centrifuge tubes, homogenizer or sonicator.
(2) 37 °C constant temperature incubator or water bath, microplate reader.
(3) Absolute ethanol, sterile deionized water, PBS.
2. Reagent Preparation
(1) Preparation of cofactor working solution: Completely dissolve 8.4 mg of cofactor powder in 1 mL of sterile deionized water to obtain a final concentration of 10 mM. It is recommended to aliquot the solution into small tubes before use, store at −20 °C protected from light, and avoid repeated freeze–thaw cycles.
(2) Preparation of inhibitor working solution: Mix 1 volume of inhibitor with 2 volumes of absolute ethanol thoroughly. Store at −20 °C protected from light.
(3) Thaw glutathione reductase on ice. Equilibrate the detection buffer, extraction buffer, and chromogenic solution to room temperature before use.
(4) Preparation of standard solution: Dilute 10 μL of GSSG standard (1 μM) with 90 μL of Extraction Buffer to prepare a 100 μM working solution. Further serially dilute the solution with Extraction Buffer to obtain concentrations of 40 μM, 20 μM, 10 μM, 5 μM, 2.5 μM, 1.25 μM, 0.625 μM and 0 μM. It is recommended to prepare a fresh standard curve for each experiment.
3. Sample Preparation for Determination of Total Glutathione Content
(1) Tissue samples: Use fresh tissue samples for detection whenever possible. Add 100–200 μL of Extraction Buffer per 10 mg of tissue, and perform rapid homogenization on ice (pre‑cool the homogenizer on ice in advance). Centrifuge the homogenized sample at 8,000 g and 4 °C for 10 min, transfer 20 μL of the supernatant for subsequent assay.
(2) Blood samples: For serum samples, allow whole blood to stand at room temperature for 30 minutes to 2 hours, then centrifuge at 1,000–2,000 g and 4 °C for 10 minutes and collect the supernatant. For plasma samples, anticoagulate whole blood with heparin or EDTA, centrifuge at 1,000–2,000 g and 4 °C for 10 minutes, transfer 20 μL of the supernatant for subsequent assay.
(3) Cell/bacterial samples: Use fresh cells or bacteria for detection; frozen samples are not recommended. Collect cells or bacteria and wash twice with pre-cooled PBS (resuspend in PBS and centrifuge at 500 g and 4 °C for 5 min). Resuspend 1×10⁶ cells or bacteria in 100–200 μL of Extraction Buffer, and lyse by ultrasonic disruption on ice (200 W, 2 s ultrasound, 3 s interval, 5 min total duration). Centrifuge the lysate at 8,000 g and 4 °C for 5 min, transfer 20 μL supernatant and keep on ice until assay.
4. Sample Preparation for Determination of GSSG Content
(1) Transfer an aliquot of the total glutathione test sample prepared above into a 1.5 mL EP tube. Add 2 μL inhibitor working solution per 28 μL sample, mix thoroughly, then incubate at 37 °C in the dark for 15 min. After centrifugation, collect 20 μL supernatant and store on ice until testing.
5. Determination of Samples and Standards
(1) Pipette samples or standard solutions sequentially into a 96-well plate following the table below.
Reagent | Blank well (μL) | Sample well (μL) | Standard well (μL) |
Sample | - | 20 | - |
Deionized water | 20 | - | - |
Standard solution | - | - | 20 |
Assay buffer | 133 | 133 | 133 |
GR | 2 | 2 | 2 |
Cofactor Working Solution | 5 | 5 | 5 |
Chromogen | 40 | 40 | 40 |
(2) Mix thoroughly, incubate at 37 °C in the dark for 10 min, then record absorbance at 412 nm.
Δstandard=Asample - Ablank；ΔStandard=Astandard - Ablank
6. Calculation of Results
(1) Standard curve: y = 0.0617*X − 0.0134 (for reference only); X represents GSSG concentration (μM), and y refers to ΔA.
(2) The concentrations of total glutathione (C) and GSSG (C) in samples can be separately calculated based on the standard curve using the formula: C (μM)=A×n
Note: A refers to the concentration (μM) calculated from the standard curve; n represents the sample dilution factor.
(3) The concentration of GSH can be calculated from the measured total glutathione and GSSG concentrations using the formula: GSH=(Total Glutathione-GSSG)×2. One molecule of GSSG can be reduced to two molecules of GSH, hence the multiplication factor of 2. For example, if the standard curve yields a total glutathione concentration of 7.5 μM and a GSSG concentration of 1.2 μM, the GSH concentration is calculated as: (7.5-1.2)×2=12.6 μM.
Precautions
1. Aliquot the prepared cofactor working solution and store at −20 °C protected from light. The inhibitor, GSSG standard and chromogenic solution shall also be stored away from light.
2. The inhibitor is toxic; all related operations should be performed in a fume hood.
3. The inhibitor used to block endogenous GSH must be added in 1.5 mL EP tubes rather than directly into the 96-well plate. The inhibitor may corrode the plate and reduce masking efficiency.
4. This product is for research use only by qualified professionals. It shall not be used for clinical diagnosis or treatment, food or pharmaceutical manufacturing, and shall not be stored in residential premises.
ZJ26F0838760
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