BioReagent 级 ，适用于对基线干扰要求严格的色谱和分析工作流程。
2-8°C储存,避光。低温运输 。请查阅批次 COA 获取详细规格。
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。
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），2-硝基-5-巯基苯甲酸在 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。
检测范围：2-400µg/mL
灵敏度：2 μg/mL
适用样本：血清（浆）、动植物组织、血细胞、细胞、细菌
R1492762 | 组件 | 物料外观 | 48T | 96T | 储存 | 单次测试使用量 |
R1492762A | 提取液 | 无色液体 | 70 mL | 70mL×2 mL | 2-8℃ | 按需 |
R1492762B | 检测缓冲液 | 无色液体 | 10 mL | 20 mL | 2-8℃. | 140 µL |
R1492762C | 显色液 | 微黄液体 | 4 mL | 8 mL | 2-8℃.避光. | 40 µL |
R1492762D | 标准品 | 粉末 | 1EA | 2EA | 2-8℃.避光. | 按需 |
请在实验前检查各组分的量。
各组分在规格基础上额外提供10%的量用于进行标准曲线制作或预实验。
自备仪器和耗材
| 自备资源类型 | 资源名称 | 备注 |
| 仪器 | 酶标仪 | 能测412nm处的吸光度 |
| 耗材 | 96 孔酶标板 | 普通透明板 |
| 试剂 | PBS（pH7.4）、去离子水 | 标准品及样本稀释用 |
| 其他 | 匀浆器（组织样本）、恒温箱、制冰机、低温离心机、 可调节式移液枪及枪头 | 检测量较大时合理使用多通道移液器有助于提高实验效率 |
实验流程
1、试剂准备
| 试剂名称 | 试剂准备 | 注意事项 |
| Extraction Buffer | 即用型；使用前平衡到室温 | 4℃保存 |
| Assay Buffer | 即用型；使用前平衡到室温 | 4℃保存 |
| Chromogen | 即用型 | 实验过程中避光放置，4℃避光保存 |
| Standard | 即用型；使用前平衡到室温 | 质量为10mg，分装避光保存于-20℃ |
2、标准品配制
使用1mL去离子水将质量为10mg的标准品溶解为浓度10mg/mL的储备液，混合均匀；平衡到室温；分装于-20℃避光保存1个月。取200µL Extraction Buffer加入1800µL去离子水配成标准品稀释液。使用标准品稀释液进一步将储备液配制为终浓度为400µg/mL、200µg/mL、100µg/mL、50µg/mL、25µg/mL、12.5 µg/mL、6.25 µg/mL 的标准品工作液。
| 标准品工作液 | 标准品（μL） | 标准品稀释液（μL） | 浓度（µg/mL） |
| 1 | 20μL of 10mg/mL | 480 | 400 |
| 2 | 100μL of 400µg/mL | 100 | 200 |
| 3 | 100μL of 200µg/mL | 100 | 100 |
| 4 | 100μL of 100µg/mL | 100 | 50 |
| 5 | 100μL of 50µg/mL | 100 | 25 |
| 6 | 100μL of 25µg/mL | 100 | 12.5 |
| 7 | 100μL of 12.5µg/mL | 100 | 6.25 |
3、样本制备
注意：推荐使用新鲜样本，如果不立即进行实验，样本可在-80℃保存1个月。处理好的样本须当天检测。测定过程中样本在冰上放置，以免变性和失活。
3.1 动物组织：
称取约0.1g动物组织样本，加入1mL预冷的Extraction Buffer，冰浴匀浆，8000g，4℃离心10min，取上清液于新离心管中，置冰上待检测。
3.2 植物组织：
称取约0.1g植物组织样本，加入1mL Extraction Buffer，冰浴超声波破碎5min（功率30%或200W，超声3s，间隔7s，重复30次），然后8000g，4℃离心10min，取上清液，置冰上待测。
3.3 细胞或细菌：
收集5×10⁶个细胞或细菌，用冷的PBS清洗细胞或细菌，800g离心2min后弃上清，加入1mL Extraction Buffer，冰浴超声波破碎5min（功率30%或200W，超声3s，间隔7s，重复30次），然后8000g，4℃离心10min，取上清液，置冰上待测。
3.4 血清或血浆：
将收集的抗凝血于600g，4℃离心10min，30min内吸取上层血浆到另一支试管中，加入等体积的Extraction Buffer，8000g，4℃离心10min，取上清液，置冰上待测。
3.5 血细胞：将收集的抗凝血于600g，4℃离心10min，弃去上层血浆用3倍体积的PBS清洗3次（用PBS重悬血细胞，600g，4℃离心10min），加入等体积Extraction Buffer，混匀后4℃放置10min，8000g，4℃离心10min，取上清液，置冰上待测。
4、实验步骤
4.1 酶标仪准备：预热30min以上，调节波长到412nm。
4.2 检测体系配制：在96孔板中按照如下方式操作，标准曲线和空白孔一般只做1次。
| 试剂 | 空白孔（μL） | 标准孔（μL） | 测定孔（μL） |
| 样本 | 0 | 0 | 20 |
| 去离子水 | 20 | 0 | 0 |
| 标准品工作液 | 0 | 20 | 0 |
| Assay Buffer | 140 | 140 | 140 |
| Chromogen | 40 | 40 | 40 |
4.3 充分混匀，反应体系在室温下避光孵育2min。
4.4 吸光度检测：在412nm处读取吸光度值，分别标记为A 空白 、A 标准 和A 测定 。
5、结果计算
以下为您提供的推导过程计算公式和简洁计算公式，两者完全相等。
5.1 数据处理
计算ΔA 标准 =A 标准 -A 空白 ，ΔA 测定 =A 测定 -A 空白 。
5.2 标准曲线绘制
以标准品浓度为y轴，ΔA 标准 为x轴，绘制标准曲线。将ΔA 测定 代入方程得到y值（µg/mL）。
5.3 样本GSH含量计算
(1) 按样本质量计算
GSH含量 (µg/g 鲜重)=y×V 样 ÷(W× V样 ÷V 样总 )×n=y÷W×n
(2) 按细胞或细菌数量计算
GSH含量 (µg/ 10⁴)=y÷(500÷V 样总 )×n=y÷500×n
(3) 按液体体积计算
GSH含量 (µg/mL)=y×2×n
(4) 按蛋白浓度数量计算
GSH含量 (µg/mg prot)=y×V 样 ÷(V 样 ×Cpr)×n=y÷Cpr×n
参数说明
V 样 ：加入样本，20µL；
n：样品稀释倍数；
Cpr：样本蛋白质浓度，mg/mL；
W：样品质量，g；
V 样总 ：样本提取体积，1mL；
500：细胞或细菌数量，以10⁴为单位；
2：提取液体时稀释一倍。
6、结果展示
典型标准曲线：y=342.21x+1.4327，R²=0.995
示例-1: 取20μL小鼠血浆，按照测定步骤操作，用96孔板进行检测。测得ΔA 测定 =A 测定 -A 空白 =0.155-0.084=0.071，代入标准曲线，计算y=25.73。按液体体积计算得：GSH含量 (µg/mL)=y×2×n =51.46 µg/mL。
注意事项
1、正式检测前，建议选择2-3个预期差异较大的样本进行预实验。
2、该试剂盒提取的样本也适用于氧化型谷胱甘肽的检测。因提取液中含有蛋白质沉淀剂，因此上清液不能用于蛋白浓度测定。如需测定蛋白含量，需另取相同样本，把提取液换成去离子水提取制备。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。
3、本试剂盒兼容分光光度计检测，请按照分光光度计检测要求按比例调节检测试剂配制量。
4、建议实验者自行建立标准曲线以提高结果准确度。如未自行建立标准曲线，可参考结果展示部分的典型标准曲线公式进行结果计算。
5、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。
6、本产品仅供科学研究使用，不适用于临床诊断。
常见问题及解答
Q：检测得到的样本ΔA测定过高或者过低怎么办？
A：如果样本ΔA 测定 >1.2，则说明样本GSH含量太高，需用Extraction Buffer适当稀释（计算公式中乘以相应稀释倍数）。如果样本的ΔA 测定 ＜0.02，可提高样本量。
Glutathione is a tripeptide containing a γ-amide bond and a sulfhydryl group, composed of glutamate, cysteine, and glycine. It is widely found in animal and plant tissues and microorganisms. In living organisms, it helps maintain normal immune system function and has antioxidant and detoxifying effects. Glutathione exists in two forms: reduced (GSH) and oxidized (GSSG). GSH is the primary intracellular antioxidant thiol substance, playing important roles in antioxidation, protein thiol protection, and amino acid transmembrane transport. The ratio of reduced to oxidized glutathione (GSH/GSSG) can serve as a key dynamic indicator for assessing the cellular redox state.
Detection Principle: Reduced glutathione (GSH) reacts with 5,5'-Dithiobis-(2-nitrobenzoic acid) (DTNB) to produce yellow-colored 5-thio-2-nitrobenzoic acid (TNB) and glutathione disulfide (GSSG). TNB has a characteristic absorption peak at 412 nm. The GSH content can be quantified by measuring the change in absorbance.
Detection Range: 2-400 µg/mL
Sensitivity: 2 µg/mL
Applicable Samples: Serum (plasma), animal/plant tissues, blood cells, cells, bacteria.
| R1492762 | Component | 48T | 96T | Storage |
| R1492762A | Extraction Buffer | 70 mL | 70 mL×2 | 2-8℃ |
| R1492762B | Assay Buffer | 10 mL | 20 mL | 2-8℃ |
| R1492762C | Chromogen | 4 mL | 8 mL | 2-8℃. Store in the dark. |
| R1492762D | Standard | 1 EA | 2 EA | 2-8℃. Store in the dark. |
Please check the quantity of each component before the experiment.
An additional 10% of each component is provided beyond the specified volume for standard curve preparation or preliminary experiments.
User-Provided Instruments and Consumables
| Type | Name | Notes |
| Instrument | Microplate Reader | Capable of measuring absorbance at 412 nm. |
| Consumables | 96-well Microplate | Standard transparent plate. |
| Reagents | PBS (pH 7.4), Deionized Water | For standard and sample dilution. |
| Others | Homogenizer (for tissue samples), incubator, ice maker, low-temperature centrifuge, adjustable pipettes and tips | Using a multichannel pipette for large-scale detection can improve efficiency. |
Experimental Procedure
1. Reagent Preparation
| Reagent Name | Reagent Preparation | 注意事项 Precautions |
| Extraction Buffer | Ready-to-use; equilibrate to room temperature before use. | Store at 4°C. |
| Assay Buffer | Ready-to-use; equilibrate to room temperature before use. | Store at 4°C. |
| Chromogen | Ready-to-use | Protect from light during the experiment; store at 4°C in the dark. |
| Standard | Ready-to-use; equilibrate to room temperature before use. | Mass is 10 mg; aliquot and store at -20°C protected from light. |
2. Standard Preparation
2.1 Dissolve the 10 mg standard in 1 mL of deionized water to prepare a 10 mg/mL stock solution. Mix well, equilibrate to room temperature. Aliquot and store at -20°C protected from light for up to 1 month.
2.2 Add 200 µL of Extraction Buffer to 1800 µL of deionized water to prepare the standard dilution buffer.
2.3 Use the standard dilution buffer to further dilute the stock solution to prepare standard working solutions with final concentrations of 400, 200, 100, 50, 25, 12.5, and 6.25 µg/mL.
| Standard Working Solution | Standard (μL) | Standard Dilution Buffer (μL) | Concentration (µg/mL) |
| 1 | 20 µL of 10 mg/mL stock | 480 | 400 |
| 2 | 100 µL of 400 µg/mL sol (from 1) | 100 | 200 |
| 3 | 100 µL of 200 µg/mL sol (from 2) | 100 | 100 |
| 4 | 100 µL of 100 µg/mL sol (from 3) | 100 | 50 |
| 5 | 100 µL of 50 µg/mL sol (from 4) | 100 | 25 |
| 6 | 100 µL of 25 µg/mL sol (from 5) | 100 | 12.5 |
| 7 | 100 µL of 12.5 µg/mL sol (from 6) | 100 | 6.25 |
3. Sample Preparation
Note: Fresh samples are recommended. If not used immediately, samples can be stored at -80°C for up to 1 month. Processed samples must be assayed on the same day. Keep samples on ice during the assay procedure to prevent denaturation and inactivation.
3.1 Animal Tissues:
Weigh approximately 0.1 g of tissue, add 1 mL of pre-cooled Extraction Buffer, and homogenize on ice. Centrifuge at 8000 g, 4°C for 10 min. Collect the supernatant into a new tube and keep on ice for detection.
3.2 Plant Tissues:
Weigh approximately 0.1 g of plant tissue, add 1 mL of Extraction Buffer, and disrupt by ultrasound on ice (power 30% or 200 W, ultrasonicate for 3 s, interval 7 s, repeat 30 times). Then centrifuge at 8000 g, 4°C for 10 min. Collect the supernatant and keep on ice for detection.
3.3 Cells or Bacteria:
Collect 5×10⁶ cells or bacteria. Wash with cold PBS, centrifuge at 800 g for 2 min, and discard the supernatant. Add 1 mL of Extraction Buffer, and disrupt by ultrasound on ice (power 30% or 200 W, ultrasonicate for 3 s, interval 7 s, repeat 30 times). Then centrifuge at 8000 g, 4°C for 10 min. Collect the supernatant and keep on ice for detection.
3.4 Serum or Plasma:
Centrifuge the collected anticoagulated blood at 600 g, 4°C for 10 min. Within 30 minutes, aspirate the upper plasma into another tube. Add an equal volume of Extraction Buffer, mix, then centrifuge at 8000 g, 4°C for 10 min. Collect the supernatant and keep on ice for detection.
3.5 Blood Cells:
Centrifuge the collected anticoagulated blood at 600 g, 4°C for 10 min. Discard the upper plasma and wash the blood cells three times with 3 volumes of PBS (resuspend blood cells in PBS, centrifuge at 600 g, 4°C for 10 min). Add an equal volume of Extraction Buffer, mix, and let stand at 4°C for 10 min. Centrifuge at 8000 g, 4°C for 10 min. Collect the supernatant and keep on ice for detection.
4. Assay Steps
4.1 Microplate Reader Preparation: Preheat for at least 30 minutes, set wavelength to 412 nm.
4.2 Assay System Setup: Perform the following operations in a 96-well plate. The standard curve and blank generally need to be performed only once.
| Reagent | Blank Well (μL) | Standard Well (μL) | Test Well (μL) |
| Sample | 0 | 0 | 20 |
| Deionized Water | 20 | 0 | 0 |
| Standard Working Solution | 0 | 20 | 0 |
| Assay Buffer | 140 | 140 | 140 |
| Chromogen | 40 | 40 | 40 |
4.3 Mix thoroughly. Incubate the reaction system at room temperature protected from light for 2 minutes.
4.4 Absorbance Measurement: Read the absorbance at 412 nm, recorded as A blank , A standard , and A test .
5. Result Calculation
The following provides both the derived formula and the simplified calculation formula, which are completely equivalent.
5.1 Data Processing
Calculate ΔA standard = A standard - A blank , ΔA test = A test - A blank .
5.2 Standard Curve Plotting
Plot the standard curve with standard concentration as the y-axis and ΔA standard as the x-axis. Substitute ΔA test into the equation to obtain the y value (µg/mL).
5.3 Sample GSH Content Calculation
(1) Based on sample mass:
GSH Content (µg/g fresh weight) = y × V sample ÷ (W × V sample ÷ V total ) × n = y ÷ W × n
(2) Based on cell or bacterial count:
GSH Content (µg/10⁴ cells) = y ÷ (500 ÷ V total ) × n = y ÷ 500 × n
(3) Based on liquid volume:
GSH Content (µg/mL) = y × 2 × n
(4) Based on protein concentration:
GSH Content (µg/mg prot) = y × V sample ÷ (V sample × Cpr) × n = y ÷ Cpr × n
Parameter Description:
V sample : Sample volume added, 20 µL;
n: Sample dilution factor;
Cpr: Sample protein concentration, mg/mL;
W: Sample mass, g;
V total : Total volume of sample extract, 1 mL;
500: Cell or bacterial count, in units of 10⁴;
2: Dilution factor (for liquid samples, diluted 1:1 with Extraction Buffer).
6. Result Presentation
Typical Standard Curve: y = 342.21x + 1.4327, R² = 0.995
Example-1: 20 μL of mouse plasma was taken and assayed according to the procedure using a 96-well plate. The measured ΔA test = A test - A blank = 0.155 - 0.084 = 0.071. Substituting into the standard curve, y = 25.73 µg/mL. Calculated based on liquid volume: GSH Content (µg/mL) = y × 2 × n = 51.46 µg/mL.
Precautions
1. It is recommended to perform preliminary experiments using 2-3 samples expected to have significant differences before formal testing.
2. The samples extracted with this kit are also suitable for the detection of oxidized glutathione (GSSG). Because the extraction buffer contains a protein precipitant, the supernatant cannot be used for protein concentration determination. If protein content needs to be measured, prepare another identical sample using deionized water instead of the extraction buffer. For protein concentration determination, Aladdin BCA Protein Quantification Kit (B665595) or Ready-to-Use BCA Protein Quantification Kit (R1491648) are recommended.
3. This kit is compatible with spectrophotometer detection. Adjust the preparation volume of detection reagents proportionally according to the spectrophotometer's requirements.
4. It is recommended to establish your own standard curve for improved accuracy. If not, you may refer to the typical standard curve formula provided in the results section for calculation.
5. Biochemical reagents are generally irritating and biologically toxic. For your safety and health, please wear appropriate personal protective equipment (lab coat, mask, gloves, hair cap, etc.) throughout the experiment and perform experiments in a fume hood or biosafety cabinet.
6. This product is for scientific research use only. Not intended for clinical diagnosis.
Frequently Asked Questions
Q: What should I do if the sample ΔA test is too high or too low?
A: If the sample ΔA test is >1.2, the GSH content in the sample is too high. Dilute the sample appropriately with Extraction Buffer (multiply by the dilution factor in the calculation). If the sample ΔA test is <0.02, increase the sample amount.
R1492762 | 组件 | 物料外观 | 48T | 96T | 储存 | 单次测试使用量 |
R1492762A | 提取液 | 无色液体 | 70 mL | 70mL×2 mL | 2-8℃ | 按需 |
R1492762B | 检测缓冲液 | 无色液体 | 10 mL | 20 mL | 2-8℃. | 140 µL |
R1492762C | 显色液 | 微黄液体 | 4 mL | 8 mL | 2-8℃.避光 | 40 µL |
R1492762D | 标准品 | 粉末 | 1EA | 2EA | 2-8℃.避光 | 按需 |
通过匹配包装上的批号来查找并下载产品的 COA，每批产品都进行了严格的验证，您可放心使用！
| 批号(Lot Number) | 证书类型 | 货号 |
|---|---|---|
 ZJ26F0737708 |
分析证书 | R1492762 |
| ZJ26F0737709 |
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| ZJ26F0737702 |
分析证书 | R1492762 |
| ZJ26F0737701 |
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| ZJ26F0333026 |
分析证书 | R1492762 |
| ZJ25F1028966 |
分析证书 | R1492762 |
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