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。
谷胱甘肽还原酶（Glutathione reductases, GR）（EC 1.6.4.2）是广泛存在于真核和原核生物中的一种黄素蛋白氧化还原酶，GR 催化 GSSG 还原生成 GSH，是谷胱甘肽氧化还原循环的关键酶之一（通常昆虫中GR被硫氧还蛋白还原酶（TrxR）取代）。GR 催化 NADPH 还原 GSSG 生成 GSH，有助于维持体内GSH/GSSG比值平衡。GR 在氧化胁迫反应中对活性氧清除起关键作用，此外，GR 还参与抗坏血酸-谷胱甘肽循环途径。
检测原理：GR 能催化 NADPH 还原 GSSG 再生 GSH，同时 NADPH 脱氢生成NADP⁺；NADPH在 340 nm 有特征吸收峰，相反NADP⁺在该波长无吸收峰；通过测定 340 nm 吸光度下降速率来测定NADPH脱氢速率，从而计算 GR 活性。
适用样本：动植物组织、细胞、细菌、血清（浆）
| G1506769 | Component | 96 T | Storage |
| G1506769A | Assay Buffer | 70 mL×2 | 2-8℃ |
| G1506769B | Substrate | 2 mL | 2-8℃. Store in the dark. |
| G1506769C | GR Cofactor | 1 EA | -20℃. Store in the dark. |
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。
自备仪器和试剂
| 自备资源类型 | 资源名称 | 备注 |
| 仪器 | 酶标仪 | 能测340 nm处的吸光度 |
| 耗材 | 96 孔酶标板 | UV 板 |
| 试剂 | PBS/去离子水 | 样本清洗用/试剂配制用 |
| 其他 | 匀浆器（组织样本）、恒温箱、冰盒、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 |
| Assay Buffer | 即用型；使用前平衡到室温 | 4℃保存 |
| Substrate | 即用型；使用前平衡到室温 | 4℃避光保存 |
| GR Cofactor | 临用前配制；每管加入2.4mL去离子水，混匀，现配现用 | 实验过程中避光冰上放置；用不完的试剂-20℃分装避光保存1个月，禁止反复冻融 |
2、样本制备
注意：
①、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。
②、样品处理等过程均需要在冰上进行，且须在当日测定酶活力，匀浆液避免反复冻融。
③、细胞中GR活性测定时，细胞数目须在3-5×10⁶之间，细胞中 GR 的提取时可加Assay Buffer后研磨或超声波处理，不能用细胞裂解液处理细胞。
④、Assay Buffer 含有一定浓度的蛋白（约1mg/mL），如需测定蛋白浓度要减去Assay Buffer本身的蛋白含量。
2.1 动植物组织
称取约0.1g样本，加入1mL预冷的Assay Buffer，冰浴匀浆，10000g，4℃离心10min，取上清置冰上待测。
2.2 细胞或细菌
收集5×10⁶个细胞或细菌，用预冷的PBS清洗细胞或细菌，800g离心2min，弃上清。加入1mL预冷的Assay Buffer进行冰浴超声破碎。超声波破碎5min（功率20%或200W，超声3s，间隔7s，重复30次），10000g，4℃离心10min，取上清置于冰上待测。
2.3 血清（浆）
直接测定。
3、实验步骤
3.1 酶标仪准备：预热30min以上，调节波长到340nm。
3.2 检测前，Assay Buffer 置于25℃（普通物种）或者37℃（哺乳动物）中预热30min以上。
3.3 检测体系配制：空白孔只需做1个即可。确保180s内吸光值变化呈线性，哺乳动物组织一般须用Assay Buffer稀释2-5倍。因通过反应速率计算酶活，使用96孔 UV板时请根据操作速度控制一次测定的样本数。在96孔 UV板中按照如下方式加样：
| 试剂（μL） | 空白孔 | 测定孔 |
| 样本 | 0 | 20 |
| Assay Buffer | 170 | 150 |
| Substrate | 10 | 10 |
| GR Cofactor | 20 | 20 |
3.4 吸光度检测
迅速混匀，立即测定340nm处吸光值。记录第10s和190s时吸光值，分别记录为A1和A2。测定孔记为A测定，空白孔记为A空白。
4、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。
4.1 数据处理
计算ΔA=A1-A2，ΔA测定=A1测定-A2测定，ΔA空白=A1空白-A2空白，ΔΔA=ΔA测定-ΔA空白
4.2 样本 GR 活性计算
(1) 按蛋白浓度计算
活性单位定义：在25℃或37℃，pH 8.0 条件下，每毫克蛋白每分钟催化 1 µmol NADPH氧化。
GR 酶活 (U/mg prot)=[ΔΔA÷(ε×d)×V反总×10⁶]÷(Cpr×V样)÷T=1.072×ΔΔA÷Cpr
(2) 按样本鲜重计算
活性单位定义：在25℃或37℃，pH 8.0 条件下，每克样品每分钟催化 1 µmol NADPH氧化。
GR 酶活 (U/g)=[ΔΔA÷(ε×d)×V反总×10⁶]÷(V样÷V样总×W)÷T=1.072×ΔΔA÷W
(3) 按细胞或细菌数量计算
活性单位定义：在25℃或37℃，pH 8.0 条件下，每10⁴个细胞或细菌每分钟催化1 µmol NADPH氧化。
GR 酶活 (U/10⁴)=[ΔΔA÷(ε×d)×V反总×10⁶]÷(N×V样÷V样总)÷T=1.072×ΔΔA÷N
(4) 按液体体积计算
活性单位定义：在25℃或37℃，pH 8.0 条件下，每毫升液体每分钟催化 1 µmol NADPH氧化。
GR 酶活 (U/mL)=[ΔΔA÷(ε×d)×V反总×10⁶]÷V样÷T=1.072×ΔΔA
参数说明：
ε：NADPH 摩尔消光系数，6.22×10³ L/mol/cm；
d：96孔 UV板光径，0.5 cm；
V反总：反应体系总体积，200μL=2×10⁻⁴ L；
10⁶：单位换算系数，1 mol=1×10⁶ μmol；
Cpr：上清液蛋白浓度，mg/mL；
W：样品质量，g；
V样：加入反应体系中上清液体积，20 μL=2×10⁻² mL；
V样总：提取液体积，1 mL； T：反应时间，3 min；
N：细胞或细菌数量，以10⁴为单位（例如细胞数量为 5×10⁶，N=500）。
5、结果展示
取0.1g小鼠肝脏，按照测定步骤操作，用 96孔 UV板进行检测。测得ΔA测定=A1测定-A2测定=0.664-0.487=0.177，ΔA空白=A1空白-A2空白=0.288-0.286=0.002，ΔΔA=ΔA测定-ΔA空白=0.177-0.002=0.175。按样本质量计算得：GR 酶活 (U/g)=1.072×ΔΔA÷W=1.876 U/g。
常见问题及解答
Q：检测得到的样本ΔΔA过高或者过低怎么办？
A：如果ΔΔA小于0.02可适当加大样本量。如果ΔΔA大于1.0，样本可用 Assay Buffer 进一步稀释，或减少提取用样本量，再重新进行测定。
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、对于组织样本及细胞样本等，可通过测定蛋白浓度来进行样本间结果均一化。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、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。
5、本产品仅供科学研究使用，不适用于临床诊断。
Glutathione reductase (GR) (EC 1.6.4.2) is a flavoprotein oxidoreductase widely found in eukaryotes and prokaryotes. GR catalyzes the reduction of glutathione disulfide (GSSG) to regenerate glutathione (GSH), making it a key enzyme in the glutathione redox cycle (typically, GR in insects is replaced by thioredoxin reductase, TrxR). GR catalyzes the NADPH-dependent reduction of GSSG to GSH, helping to maintain the balance of the GSH/GSSG ratio in vivo. GR plays a critical role in scavenging reactive oxygen species under oxidative stress. Additionally, GR is involved in the ascorbate-glutathione cycle pathway.
Detection Principle
GR catalyzes the NADPH-dependent reduction of GSSG to regenerate GSH, simultaneously dehydrogenating NADPH to generate NADP⁺. NADPH has a characteristic absorption peak at 340 nm, whereas NADP⁺ does not absorb at this wavelength. The GR activity is determined by measuring the rate of decrease in absorbance at 340 nm, which corresponds to the rate of NADPH dehydrogenation.
Applicable Samples: Animal and plant tissues, cells, bacteria, serum (plasma).
| G1506769 | Component | 96 T | Storage |
| G1506769A | Assay Buffer | 70 mL×2 | 2-8℃ |
| G1506769B | Substrate | 2 mL | 2-8℃. Store in the dark. |
| G1506769C | GR Cofactor | 1 EA | -20℃. Store in the dark. |
Please verify the quantity of all components before commencing the experiment.
An additional 10% of each component is supplied beyond the specified volume/amount to facilitate standard curve preparation or preliminary testing.
Materials Required but Not Provided
| Category | Resource Name | Notes |
| Instrument | Microplate Reader | Capable of measuring absorbance at 340 nm |
| Consumables | 96-well Microplate | UV-transparent plate |
| Reagents | PBS / Deionized Water | For sample washing / reagent preparation |
| Others | Homogenizer (for tissue samples), Incubator, Ice Bucket, Refrigerated Centrifuge, Adjustable Pipettes and Tips | For high-throughput detection, using a multichannel pipette can improve efficiency. |
Experimental Procedure
1. Reagent Preparation
| Reagent | Preparation | Notes |
| Assay Buffer | Ready-to-use; equilibrate to room temperature before use. | Store at 4°C. |
| Substrate | Ready-to-use; equilibrate to room temperature before use. | Store at 4°C protected from light. |
| GR Cofactor | Prepare immediately before use. Add 2.4 mL deionized water to each vial and mix thoroughly. | Keep protected from light on ice during the experiment. Unused reagent can be aliquoted and stored at -20°C protected from light for 1 month; avoid repeated freeze-thaw cycles. |
2. Sample Preparation
Notes:
① Fresh samples are recommended. If not assayed immediately, samples can be stored at -80°C for up to 1 month.
② All sample processing steps should be performed on ice. Enzyme activity should be measured on the same day. Avoid repeated freeze-thaw cycles of the homogenate.
③ For GR activity measurement in cells, the cell number should be between 3-5×10⁶. Use Assay Buffer for homogenization or ultrasonic disruption during GR extraction from cells; do not use cell lysis buffer.
④ Assay Buffer contains a certain concentration of protein (approx. 1 mg/mL). If determining protein concentration, subtract the protein content of the Assay Buffer itself.
2.1 Animal/Plant Tissues
Weigh approximately 0.1 g of sample, add 1 mL of pre-cooled Assay Buffer, and homogenize in an ice bath. Centrifuge at 10,000 × g, 4°C for 10 minutes. Collect the supernatant and place it on ice for assay.
2.2 Cells or Bacteria
Collect 5×10⁶ cells or bacteria. Wash with pre-cooled PBS, centrifuge at 800 × g for 2 minutes, and discard the supernatant. Add 1 mL of pre-cooled Assay Buffer and disrupt using an ultrasonic cell disruptor on ice. Ultrasonicate for 5 minutes (20% power or 200 W, pulse for 3 s, pause for 7 s, repeat 30 times). Centrifuge at 10,000 × g, 4°C for 10 minutes. Collect the supernatant and place it on ice for assay.
2.3 Serum (Plasma)
Assay directly.
3.. Assay Procedure
3.1 Microplate Reader Preparation: Preheat for at least 30 minutes. Set the wavelength to 340 nm.
3.2 Reagent Pre-incubation: Pre-warm the Assay Buffer at 25°C (for common species) or 37°C (for mammals) for at least 30 minutes before the assay.
3.3 Assay Setup: One blank well is sufficient. Ensure the absorbance change is linear within 180s. Mammalian tissues generally need to be diluted 2-5 times with Assay Buffer. As enzyme activity is calculated from the reaction rate, control the number of samples measured at once based on operation speed when using a 96-well UV plate. Add reagents to the 96-well UV plate as follows:
| Reagent (μL) | Blank Well | Test Well |
| Sample | 0 | 20 |
| Assay Buffer | 170 | 150 |
| Substrate | 10 | 10 |
| GR Cofactor | 20 | 20 |
3.4 Absorbance Measurement:
Mix rapidly immediately after adding the GR Cofactor. Measure the absorbance at 340 nm. Record the absorbance at 10 seconds as A1 and at 190 seconds as A2. For the Test well, record as A1test and A2test; for the Blank well, record as A1blank and A2blank.
4. Calculation of Results
The following provides both the detailed derivation formula and the simplified formula. They are exactly equivalent.
4.1 Data Processing
Calculate ΔA = A1 - A2 for each well.
Frequently Asked Questions
Q: What should I do if the measured ΔΔA for the sample is too high or too low?
A: If ΔΔA is less than 0.02, consider appropriately increasing the sample amount. If ΔΔA is greater than 1.0, the sample can be further diluted with Assay Buffer, or the sample amount used for extraction can be reduced, before re-assaying.
Precautions
Before formal testing, it is recommended to perform preliminary assays using 2-3 samples expected to have significant differences.
For tissue and cell samples, results can be normalized by measuring protein concentration. The Aladdin B665595 BCA Protein Quantification Kit or R1491648 Ready-to-Use BCA Protein Quantification Kit is recommended.
This kit is compatible with spectrophotometer detection. Adjust the preparation volume of detection reagents proportionally according to the spectrophotometer's requirements.
Biochemical reagents are generally irritating and biologically toxic. For your safety and health, implement appropriate biosafety precautions throughout the experiment, including wearing lab coats, masks, gloves, head covers, etc., and perform experiments in a fume hood or biosafety cabinet.
This product is for research use only. Not for use in diagnostic procedures.
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 |
|---|---|---|
 ZJ25F1230305 |
分析证书 | G1506769 |
| I2604227 |
分析证书 | G1506769 |
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