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S-转移酶（GST）是一种具有多种生理功能的蛋白质家族，主要存在于细胞质内。GST是体内解毒酶系统的重要组成部分，主要催化各种化学物质及其代谢产物与GSH的巯基共价结合，使亲电化合物变为亲水化合物，易于从胆汁或尿液中排泄，达到将体内各种潜在或具有毒性的物质降解并排出体外的目的。因此，GST在保护细胞免受亲电化合物的损伤中发挥着重要的生物学功能。GST具有GSH-Px活性，亦称为non-SeGSH-Px，具有修复氧化破坏的大分子如DNA、蛋白质等的功能。GST催化反应会减少GSH含量，但是不增加GSSG含量。
检测原理：GST催化GSH与CDNB结合，其结合产物的光吸收峰波长为340nm；通过测定340nm波长处吸光度上升速率，即可计算出GST活性。
检测范围：2-76 U/L
灵敏度：2 U/L
适用样本：血清（浆）、动植物组织、细胞、细菌
| G1501773 | Component | 48T | 96T | Storage |
| G1501773A | Assay Buffer | 60 mL | 120 mL | 2-8℃ |
| G1501773B | Chromogen | 11 mL | 22 mL | 2-8℃. Store in the dark. |
| G1501773C | Substrate | 1EA | 1EA | 2-8℃. Store in the dark. |
注意：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。
自备仪器和试剂
酶标仪或紫外分光光度计（能测340nm处的吸光度）
96孔UV板或微量石英比色皿、可调节式移液枪及枪头
制冰机、低温离心机、水浴锅
去离子水
匀浆器（如果是组织样本）
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 |
| Chromogen | 即用型；使用前平衡到室温 | 4℃避光保存；对皮肤有刺激性，建议操作时做好防护。 |
| Substrate Working Reagent | 使用前加入2.4mL离子水溶解 | 用不完的试剂4℃避光保存可稳定1个月 |
2、样本准备
2.1 动植物组织
称取0.1g组织样本，加入1mL预冷的Assay Buffer，快速冰上匀浆。匀浆后的样本，8,000g，4℃离心10min，取上清液，置冰上待测。
2.2 细胞或细菌
收集5×10⁶细胞或细菌到离心管内，用冷PBS清洗细胞，离心后弃上清，加入1mL Assay Buffer，冰浴超声波破碎细胞或细菌5min（功率20%或200W，超声3s，间隔7s，重复30次），然后8,000g，4℃离心10min，取上清液，置冰上待测。
2.3 血清（浆）
直接测定。
注意：
样品处理等过程均需要在冰上进行，如果不立即进行实验，样本可在-80°C保存一个月。
细胞中GST活性测定时，细胞数目须在3-5×10⁶之间，细胞中GST的提取时可加Assay Buffer后超声波处理，不能用细胞裂解液处理细胞。
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、实验步骤
3.1 酶标仪或紫外分光光度计预热30min以上，调节波长到340nm，可见紫外分光光度计用去离子水调零。
3.2 Substrate Working Reagent；置于25℃（一般物种）或者37℃（哺乳动物）孵育15min。
3.3 在96孔UV板或微量石英比色皿中按照如下方式加样：
| 试剂 | 空白 (μL) | 测定 (μL) |
| Sample | 0 | 20 |
| Assay Buffer | 20 | 0 |
| Chromogen | 180 | 180 |
| Substrate Working Reagent | 20 | 20 |
3.4 迅速混匀后于340nm测定吸光度变化，分别记录空白的10s为A1，310s为A2；测定的10s为A3，310s为A4。计算ΔA 空 =A2-A1，ΔA 测 =A4-A3。
注意： 空白只需做1个。
实验之前建议选择2-3个预期差异大的样本做预实验。若样品测定吸光度大于1，建议对样品用去离子水稀释，计算时结果乘以稀释倍数。测定反应的温度对测定结果有影响，请控制在25°C（一般物种）或者37°C（哺乳动物）。
4、结果计算
注意：我们为您提供的计算公式，包括推导过程计算公式和简洁计算公式。两者完全相等。建议以加粗的简洁计算公式为最终计算公式。
4.1 使用96孔UV板测定的计算公式
(1) 按蛋白浓度计算
(1) 按蛋白浓度计算 活性单位定义：在25°C或者37°C中，每毫克蛋白每分钟催化1μmol/L CDNB与GSH结合。 GST (U/mg prot)=(ΔA 测 -ΔA 空 )÷(ε×d)×10⁶×V 反总 ÷(Cpr×V 样 )÷T=0.46×(ΔA 测 -ΔA 空 )÷Cpr
(2) 按样本鲜重计算
(2) 按样本鲜重计算 活性单位定义：在25°C或者37°C中，每克样品每分钟催化1μmol/L CDNB与GSH结合。 GST (U/g鲜重)=(ΔA 测 -ΔA 空 )÷(ε×d)×10⁶×V 反总 ÷(V 样 ÷V 样总 ×W)÷T=0.46×(ΔA 测 -ΔA 空 )÷W
(3) 按细胞或细菌数量计算
(3) 按细胞或细菌数量计算 活性单位定义：在25°C或者37°C中，每10⁴个细胞或细菌每分钟催化1μmol/L CDNB与GSH结合。 GST (U/10⁴)=(ΔA 测 -ΔA 空 )÷(ε×d)×10⁶×V 反总 ÷(500×V 样 ÷V 样总 )÷T=0.46×(ΔA 测 -ΔA 空 )÷500
(4) 按液体体积计算
(4) 按液体体积计算 活性单位定义：在25°C或者37°C中，每毫升液体每分钟催化1μmol/L CDNB与GSH结合。 GST (U/mL)=(ΔA 测 -ΔA 空 )÷(ε×d)×10⁶×V 反总 ÷V 样 ÷T=0.46×(ΔA 测 -ΔA 空 )
4.2 使用微量石英比色皿测定的计算公式
将上述计算公式中的光径d=0.5cm调整为d=1cm进行计算即可。
参数说明：
ε：产物摩尔消光系数，9.6×10³ L/mol/cm；
d：96孔板光径，0.5cm； 10⁶：1mol=1× 10⁶ μmol；
V 反总 ：反应体系总体积，220μL=2.2×10⁻⁴ L；
Cpr：上清液蛋白质浓度，mg/mL；
W：样品质量，g；
V 样 ：加入反应体系中上清液体积，20μL=0.02mL；
V 样总 ：加入提取液体积，1mL；
T：反应时间，5min；
500：细胞或细菌数量，5×10⁶。
注意事项
1、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。
2、本产品仅供科学研究使用，不适用于临床诊断。
结果展示
取 0.1 g 玉米种子，按照测定步骤操作，用 96 孔 UV 板进行检测。测得 ΔA测定=A2 测定−A1 测定=0.56−0.431 =0.129，ΔA空白=A2 空白−A1 空白=0.242−0.241=0.001，ΔΔA=ΔA测定−ΔA空白=0.129−0.001=0.128。按样本质量计算得：GST 酶活 (U/g)=0.46 ×ΔΔA÷W=0.589 U/g。
FAQ
检测得到的样本ΔΔA 过高或者过低怎么办？ 如果ΔΔA 小于0 .005 可适当加大样本量或延长反应到10-15 min。如果ΔΔA 大于1.0，样本可用去离子水进一步稀释，再重新进行测定。测定反应的温度对测定结果有影响，请控制在25℃（其它物种）或者37℃（哺乳动物）。
Glutathione S-Transferase (GST) is a protein family with multiple physiological functions, primarily located within the cytoplasm. GST is a crucial component of the body's detoxification enzyme system. It mainly catalyzes the covalent binding of the sulfhydryl group of glutathione (GSH) to various chemicals and their metabolites, converting electrophilic compounds into hydrophilic compounds that are more easily excreted in bile or urine. This process facilitates the degradation and elimination of potentially toxic substances from the body. Thus, GST plays a vital biological role in protecting cells from damage caused by electrophilic compounds. GST possesses GSH peroxidase activity (also known as non-Se-GSH-Px) and functions in repairing oxidatively damaged macromolecules such as DNA and proteins. The GST-catalyzed reaction consumes GSH but does not increase GSSG levels.
Detection Principle: GST catalyzes the conjugation of GSH with CDNB (1-chloro-2,4-dinitrobenzene). The conjugation product has an absorption peak at 340 nm. The GST activity is calculated by measuring the rate of increase in absorbance at 340 nm.
Detection Range: 2 - 76 U/L
Sensitivity: 2 U/L
Applicable Samples: Serum (plasma), animal/plant tissues, cells, bacteria
| G1501773 | Component | 48T | 96T | Storage |
| G1501773A | Assay Buffer | 60 mL | 120 mL | 2-8℃ |
| G1501773B | Chromogen | 11 mL | 22 mL | 2-8℃. Store in the dark. |
| G1501773C | Substrate | 1EA | 1EA | 2-8℃. Store in the dark. |
Note: Before formal testing, it is recommended to perform a preliminary test with 2-3 samples expected to have significant differences.
User-Prepared Instruments and Reagents
96-well UV plate or micro quartz cuvettes, adjustable micropipettes and tips
Ice maker, refrigerated centrifuge, water bath
Deionized water
Homogenizer (for tissue samples)
Experimental Procedure
1. Reagent Preparation
| Reagent Name | Reagent Preparation | Notes |
| Assay Buffer | Ready-to-use; Equilibrate to room temperature before use. | Store at 4℃. |
| Chromogen | Ready-to-use; Equilibrate to room temperature before use. | Store at 4°C protected from light. Skin irritant. Use appropriate protective equipment. |
| Substrate Working Reagent | Before use, dissolve in 2.4 mL of deionized water. | Unused reagent can be stored at 4°C protected from light for one month. |
2. Sample Preparation
2.1 Animal/Plant Tissues
Weigh 0.1 g of tissue sample. Add 1 mL of pre-cooled Assay Buffer and homogenize quickly on ice. Centrifuge the homogenate at 8,000 g, 4°C for 10 minutes. Collect the supernatant and keep it on ice for assay.
2.2 Cells or Bacteria
Collect 5×10⁶ cells or bacteria into a centrifuge tube. Wash with cold PBS, centrifuge, and discard the supernatant. Add 1 mL of Assay Buffer. Sonicate in an ice bath for 5 minutes (power 20% or 200W, pulse 3s on, 7s off, repeat 30 times). Centrifuge at 8,000 g, 4°C for 10 minutes. Collect the supernatant and keep it on ice for assay.
2.3 Serum (Plasma)
Assay directly.
Note:
Sample processing should be performed on ice. If not used immediately, samples can be stored at -80°C for one month.
For GST activity measurement in cells, the cell count should be between 3-5×10⁶. Use Assay Buffer with sonication for cell extraction; do not use cell lysis buffers.
If protein concentration measurement is required, Aladdin's BCA Protein Quantification Kit (B665595) or Ready-to-Use BCA Protein Quantification Kit (R1491648) is recommended.
3. Assay Steps
3.1 Preheat the microplate reader or UV spectrophotometer for at least 30 minutes. Set the wavelength to 340 nm. For UV spectrophotometers, zero the instrument with deionized water.
3.2 Incubate the Substrate Working Reagent at 25°C (for general species) or 37°C (for mammals) for 15 minutes.
3.3 Add reagents to a 96-well UV plate or micro quartz cuvette as follows:
| Reagent | Blank (μL) | Test (μL) |
| Sample | 0 | 20 |
| Assay Buffer | 20 | 0 |
| Chromogen | 180 | 180 |
| Substrate Working Reagent | 20 | 20 |
3.4 Mix rapidly and immediately measure the change in absorbance at 340 nm. Record the absorbance for the Blank at 10 seconds (A1) and 310 seconds (A2). Record the absorbance for the Test at 10 seconds (A3) and 310 seconds (A4). Calculate ΔA blank = A2 - A1, ΔA test = A4 - A3.
Note: Only one Blank is needed. A preliminary test with 2-3 samples showing expected significant differences is recommended. If the sample absorbance is greater than 1, dilute the sample with deionized water and multiply the result by the dilution factor. The reaction temperature significantly affects the results; maintain it at 25°C (general species) or 37°C (mammals).
4. Calculation of Results
Note: We provide both derived and simplified calculation formulas. They are equivalent. The simplified formulas in bold are recommended for final calculation.
4.1 Calculation Formulas for 96-well UV Plate
(1) Based on Protein Concentration
Unit Definition: One unit of enzyme activity is defined as the amount that catalyzes the conjugation of 1 μmol of CDNB with GSH per minute per milligram of protein at 25°C or 37°C.
Calculation Formula: GST Activity (U/mg prot) = (ΔAtest - ΔAblank) ÷ (ε × d) × 10⁶ × Vtotal reaction ÷ (Cpr × Vsample) ÷ T = 0.46 × (ΔAtest - ΔAblank) ÷ Cpr
(2) Based on Sample Fresh Weight
Unit Definition: One unit of enzyme activity is defined as the amount that catalyzes the conjugation of 1 μmol of CDNB with GSH per minute per gram of sample at 25°C or 37°C.
Calculation Formula: GST Activity (U/g fresh weight) = (ΔAtest - ΔAblank) ÷ (ε × d) × 10⁶ × Vtotal reaction ÷ (Vsample ÷ Vtotal sample × W) ÷ T = 0.46 × (ΔAtest - ΔAblank) ÷ W
(3) Based on Cell or Bacterial Count
Unit Definition: One unit of enzyme activity is defined as the amount that catalyzes the conjugation of 1 μmol of CDNB with GSH per minute per 10⁴ cells or bacteria at 25°C or 37°C.
Calculation Formula: GST Activity (U/10⁴) = (ΔAtest - ΔAblank) ÷ (ε × d) × 10⁶ × Vtotal reaction ÷ (500 × Vsample ÷ Vtotal sample) ÷ T = 0.46 × (ΔAtest - ΔAblank) ÷ 500
(4) Based on Liquid Volume
Unit Definition: One unit of enzyme activity is defined as the amount that catalyzes the conjugation of 1 μmol of CDNB with GSH per minute per milliliter of liquid at 25°C or 37°C.
Calculation Formula: GST Activity (U/mL) = (ΔAtest - ΔAblank) ÷ (ε × d) × 10⁶ × Vtotal reaction ÷ Vsample ÷ T = 0.46 × (ΔAtest - ΔAblank)
4.2 Calculation Formulas for Micro Quartz Cuvette
Adjust the pathlength (d) in the formulas above from 0.5 cm to 1 cm for calculations.
Parameter Definitions:
ε: Molar extinction coefficient of the product, 9.6 × 10³ L/mol/cm
d: Light path for 96-well plate (0.5 cm) 10⁶: Conversion factor (1 mol = 1 × 10⁶ μmol)
V total reaction : Total reaction volume (220 μL = 2.2 × 10⁻⁴ L)
Cpr: Protein concentration of the supernatant (mg/mL)
W: Sample weight (g)
V sample : Volume of supernatant added to the reaction system (20 μL = 0.02 mL)
V total sample : Volume of extraction buffer added (1 mL) T: Reaction time (5 min)
500: Cell or bacterial count factor (5 × 10⁶ total cells / 10⁴ per unit = 500)
Precautions
1. Biochemical reagents are generally irritating and potentially biologically toxic. For your safety and health, implement appropriate biosafety precautions throughout the experiment, including wearing lab coats, masks, gloves, and head covers. Perform experiments in a fume hood or biosafety cabinet.
2. This product is for research use only. Not for use in clinical diagnosis.
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| 批号(Lot Number) | 证书类型 | 货号 |
|---|---|---|
 ZJ25F1129286 |
分析证书 | G1501773 |
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