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总谷胱甘肽 (T-GSH) 含量检测试剂盒 (DTNB, 微量法)
有货
为什么选择此级别
BioReagent,标曲法,比色法,适用于分析 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
-20°C储存。超低温运输 。请查阅批次 COA 获取详细规格。
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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
产品介绍
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，而GSH可以和生色底物5,5-二硫双 (2-硝基苯甲酸）(DTNB) 反应产生黄色的TNB和GSSG，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。
T1521744 | 组件 | 物理外观 | 100T | 储存 | 单次测试使用量 |
T1521744A | 检测缓冲液 | 无色液体 | 14 mL | -20℃. | 133 µL |
T1521744B | 提取液 | 无色液体 | 60 mL | -20℃. | 按需 |
T1521744C | 谷胱甘肽还原酶 | 无色液体 | 200 µL | -20℃.避光. | 2 µL |
T1521744D | 辅因子 | 粉末 | 8.4 mg | -20℃.避光. | 按需 |
T1521744E | 显色液 | 淡黄色液体 | 4 mL | -20℃.避光. | 40 µL |
T1521744F | 标准品 (GSH) | 无色液体 | 1 mL | -20℃.避光. | 按需 |
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) 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。
(3) 标准品配制：取10 μL GSH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. 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. 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。ΔA=A测定 - A空白；Δ标=A标准 - A空白;
5.结果计算
(1) 标准曲线：y=0.0327*X -0.0277(仅供参考)；x是GSH的浓度(μM)，y 是△标。
(2) 可根据标准曲线计算出样品中的总谷胱甘肽 ((T-GSH) )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GSH浓度 (μM)；n为样品稀释倍数。
注意事项
1. 辅因子配制完后记得分装-20℃避光保存，GSH标准品和显色液同样避光保存。
2. 如样品中总谷胱甘肽含量过低，可延长反应时间至20或30min。
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。
Product Introduction
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 assay kit is based on the principle that glutathione reductase (GR) reduces GSSG to GSH. GSH then reacts with the chromogenic substrate 5,5′-dithio-bis-(2-nitrobenzoic acid) (DTNB) to yield yellow TNB and GSSG. The total glutathione content in samples can be calculated by measuring the change in absorbance at 412 nm.
T1521744 | Components | Appearance | 100T | Storage | Usage volume per test |
T1521744A | Assay Buffer | Colorless liquid | 14 mL | -20℃. | 133 µL |
T1521744B | Extraction Buffer | Colorless liquid | 60 mL | -20℃. | As need |
T1521744C | GR | Colorless liquid | 200 µL | -20℃.Store in the dark. | 2 µL |
T1521744D | Cofactor | Powder | 8.4 mg | -20℃.Store in the dark. | As need |
T1521744E | Chromogen | light yellow liquid | 4 mL | -20℃.Store in the dark. | 40 µL |
T1521744F | Standard (GSH) | Colorless liquid | 1 mL | -20℃.Store in the dark. | As need |
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) 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) Thaw glutathione reductase on ice. Equilibrate the detection buffer, extraction buffer, and chromogenic solution to room temperature before use.
(3) Preparation of standard solution: Dilute 10 μL of GSH standard (1 mM) 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
(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. 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. ΔA = Asample - Ablank; ΔStandard = Astandard - Ablank..
5. Calculation of Results
(1) Standard curve: y = 0.0327*X − 0.0277 (for reference only); X represents GSH concentration (μM), and y refers to ΔStandard.
(2) The concentrations of total glutathione (C) in samples can be 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.
Precautions
1. Aliquot the prepared cofactor working solution and store at −20 °C protected from light. The GSH standard and chromogenic solution shall also be stored away from light.
2. When total glutathione concentration in samples is low, extend the reaction time to 20 or 30 min.
3. 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.
规格
规格或纯度
BioReagent, 标曲法, 适用于分析, 比色法
英文名称
Total Glutathione (T-GSH) Content Assay Kit (DTNB, Micro-method)
应用
细胞代谢
样品类型
动物组织, 植物组织, 细胞, 血浆, 血清
检测范围
0.625 μM-40 μM
检测方法
DTNB，微量法
定量&半定量
定量
检测仪器
酶标仪
检测波长
412nm
敏感性
Light-sensitive
检测时长
10 min
样本体积
20 μL
储存与运输
储存条件
-20°C储存
运输条件
超低温运输
稳定性与储存
各组分在相应的储存条件下保质期1年。
组分与储存
T1521744 | 组件 | 物理外观 | 100T | 储存 | 单次测试使用量 |
T1521744A | 检测缓冲液 | 无色液体 | 14 mL | -20℃. | 133 µL |
T1521744B | 提取液 | 无色液体 | 60 mL | -20℃. | 按需 |
T1521744C | 谷胱甘肽还原酶 | 无色液体 | 200 µL | -20℃.避光. | 2 µL |
T1521744D | 辅因子 | 粉末 | 8.4 mg | -20℃.避光. | 按需 |
T1521744E | 显色液 | 淡黄色液体 | 4 mL | -20℃.避光. | 40 µL |
T1521744F | 标准品 (GSH) | 无色液体 | 1 mL | -20℃.避光. | 按需 |
技术文档
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Establishment and Evaluation of Animal Models of Gastric Ulcer: Chemical Induction, Stress Injury, Infection Models, and Healing Studies
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