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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（Glutathione S-transferase, GST）是生物体内重要的Ⅱ相解毒酶，能催化还原型谷胱甘肽（GSH）与多种亲电子底物的结合反应。本试剂盒采用经典的CDNB/BDNB作为底物，通过检测340nm处吸光度的上升速率，即可定量分析GST的活性。
本试剂盒为微量基础版，采用粉末形式稳定技术，确保了核心试剂的长期稳定性。操作流程针对96孔板优化，具有高通量、灵敏度高、重复性好的特点，特别适用于大量样本的快速筛选与比较分析。
自备仪器与试剂
紫外可见分光光度计（带96孔板检测功能）
多通道移液器
离心机、涡旋混合器
蒸馏水、无水乙醇（若需）
蛋白浓度测定试剂盒（如Bradford法）
操作步骤
1、工作液配制 (临用前配制)
4 mM GSH 工作液: 向GSH粉末管中加入 4 mL 1×检测缓冲液，涡旋溶解。
2 mM BDNB 工作液: 向BDNB粉末粉管中加入 10mL 1×检测缓冲液，涡旋溶解，并置于避光处。
2、样本制备
组织或细胞样本：建议使用预冷的1×检测缓冲液进行匀浆或裂解。10,000g，4℃离心10分钟，取上清置于冰上待测。请用Bradford法等测定上清液的蛋白浓度。
3、加样与检测
在96孔UV板中按下表加入试剂：
| 试剂 | 测定孔 | 空白孔 |
4 mM GSH 工作液 | 25 μL | 25 μL |
| 待测样本 | 25 μL | - |
| 1× 检测缓冲液 | - | 25 μL |
| 2 mM BDNB 工作液 | 50 μL | 50 μL |
| 总体积 | 100 μL | 100 μL |
加入BDNB工作液后，请立即使用多通道移液器吹打混匀3-5次。
迅速将反应板放入已预热至37°C的酶标仪中。
立即于340nm波长下，进行动力学监测，每30秒读取一次吸光度，持续5-10分钟。
结果计算
1、计算反应速率
使用酶标仪软件，计算各孔吸光度随时间变化的线性斜率，即为 V (ΔA/min)。
校正速率 = V (测定孔) - V (空白孔)
2、计算相对GST活性
按蛋白浓度计算： 相对活性 (U/mg prot) = 校正速率 (ΔA/min) ÷ Cpr (mg/mL)
按样本质量计算：相对活性 (U/g) = 校正速率 (ΔA/min) ÷ W (g)
活性单位定义 / Unit Definition: 在37℃条件下，每毫克蛋白（或每克组织）每分钟引起340nm吸光度变化1.0定义为一个相对酶活力单位 (U)。
注意事项
1、整个样本处理过程请在冰上进行，以保持酶活性。
2、GSH和BDNB工作液溶解后，建议当天使用完毕。未用完的液体可在2-8℃避光保存1周。
3、为保证结果准确性，建议每个样本设置2-3个复孔。
4、若测定孔的初始吸光度很高（>1.5）或反应曲线非线性，请用1×检测缓冲液适当稀释样本后再测，计算结果时乘以稀释倍数。
5、本产品仅供科学研究使用。
Glutathione S-transferase (GST) is a crucial Phase II detoxification enzyme in living organisms. It catalyzes the conjugation of reduced glutathione (GSH) to a variety of electrophilic substrates. This kit uses the classic substrate CDNB/BDNB. By measuring the increasing rate of absorbance at 340 nm, the GST activity can be quantified.
This is a Basic Micro-version Kit that utilizes powder stabilization technology to ensure long-term stability of core reagents. The procedure is optimized for 96-well plates, featuring high-throughput, high sensitivity, and excellent reproducibility. It is particularly suitable for rapid screening and comparative analysis of large sample sizes.
Add reagents to a 96-well UV plate according to the table below:
Reagent | Test Well | Blank Well |
| 4 mM GSH Working Solution | 25 μL | 25 μL |
| Sample | 25 μL | - |
| 1× Assay Buffer | - | 25 μL |
| 2 mM BDNB Working Solution | 50 μL | 50 μL |
| Total Volume | 100 μL | 100 μL |
Immediately after adding the BDNB working solution, mix by pipetting up and down 3-5 times using a multichannel pipette.
Quickly place the reaction plate into a pre-warmed (37°C) microplate reader.
Immediately initiate kinetic measurement, recording the absorbance at 340 nm every 30 seconds for 5-10 minutes.
Calculation of Results
*Adjusted for Substrate Blank
**Using the extinction coefficient 9600 M-1cm-1
***Using the path correction 0.320 cm
Note: the output of many spectrophotometers is in mOD. Per Well:
1. Calculate the Reaction Rate
Use the microplate reader software to calculate the linear slope of absorbance change over time for each well, which is V (ΔA/min).
Corrected V = V (Test Well) - V (Blank Well)
2. Calculate Relative GST Activity
By Protein Concentration: Relative Activity (U/mg prot) = Corrected V (ΔA/min) ÷ Cpr (mg/mL)
By Sample Weight: Relative Activity (U/g) = Corrected V (ΔA/min) ÷ W (g)
One unit of relative enzyme activity (U) is defined as the amount of enzyme that causes a change of 1.0 absorbance unit per minute at 340 nm under the standard assay conditions (37°C) per mg of protein (or per gram of tissue).
Notes
1. Keep samples on ice during the entire preparation procedure to maintain enzyme activity.
2. It is recommended to use the reconstituted GSH and BDNB working solutions on the same day. Unused solutions can be stored at 2-8°C protected from light for up to 1 week.
3. For accurate results, it is recommended to set 2-3 replicates for each sample.
4. If the initial absorbance of the test well is too high (>1.5) or the reaction curve is non-linear, dilute the sample appropriately with 1× Assay Buffer and remeasure. Multiply the result by the dilution factor.
5. This product is for research use only.
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