本试剂盒检测原理是谷胱甘肽过氧化物酶 (GPX) 催化过氧化物氧化GSH产生GSSG，谷胱甘肽还原酶 (GR) 催化NADPH还原GSSG再生成GSH，同时NADPH被氧化成NADP⁺，测定NADPH在340nm处吸光度值的减少速率即可计算样本中GPX的活性。通过在反应体系中加入GPX4抑制剂，可测定总GPX酶活力以及GPX4之外的非特异酶活力，两者之间的差值即为GPX4酶活力。

组分与储存：

组分货号	组分名称	物理外观	48T	96T	储存
G1527167A	提取液一	液体	30 mL	60 mL	-20℃.
G1527167B	提取液二	液体	0.3 mL	0.6 mL	-20℃.
G1527167C	试剂一	液体	15 mL	30 mL	-20℃.
G1527167D	试剂二	液体	0.75 mL	1.5 mL	-20℃.
G1527167E	试剂三	液体	0.75 mL	1.5 mL	-20℃.
G1527167F	试剂四	液体	50 μL	100 μL	-20℃.
G1527167G	试剂五	液体	0.15 mL	0.3 mL	-20℃.
G1527167H	试剂六	液体	0.6 mL	1.2 mL	-20℃.
G1527167I	试剂七	液体	0.6 mL	1.2 mL	-20℃.

*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%的量用于进行预实验。

自备仪器与试剂：

自备资源类型	资源名称	备注
仪器	酶标仪	能测340nm处的吸光度
耗材	96孔酶标板	UV板
试剂	PBS、生理盐水	用于清洗样本
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	样本匀浆操作全程冰上/低温

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。推荐使用阿拉丁货号B665595 的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。

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。

实验流程：

1. 试剂准备

试剂名称	试剂准备	注意事项
提取液一	即用型；使用前平衡到室温	-20℃保存
提取液二	即用型；使用前平衡到室温	-20℃保存
工作提取液	临用前配制；每1mL的提取液一中加入10μL的提取液二	配制后当天使用有效
试剂一	即用型；使用前平衡到室温	-20℃保存
试剂二	即用型；使用前平衡到室温	-20℃保存
试剂三	即用型；使用前平衡到室温	-20℃保存
试剂四	即用型；使用前平衡到室温	-20℃保存
试剂五	临用前配制；每0.9mL的试剂一中加入0.1mL的试剂五	检测前配制，4小时内有效
试剂六	临用前配制；每0.8mL的试剂一中加入0.2mL的试剂六	检测前配制，4小时内有效
试剂七	临用前配制；每0.8mL的试剂一中加入0.2mL的试剂七	检测前配制，4小时内有效

2. 样本制备

注意：推荐使用新鲜样本，冻存样本检测酶活结果可能降低。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。

① 动物组织：用冷的PBS清洗组织，尽可能去除血液。吸干组织上的水分，称重。称取 0.1g 组织样本，加入 0.5 mL 的工作提取液，快速冰上匀浆。匀浆后的样本，12000g，4℃离心10 min，取上清液置冰上待测。组织样本建议用工作提取液稀释20倍后检测，肝脏样本建议稀释40 倍后检测。

② 细胞 (菌) 样本：收集 5×10⁶ 细胞 (菌) 到离心管内，加入 0.5mL 的工作提取液，冰浴超声波破碎细胞 (菌) 5min (功率20%或200W，超声3s，间隔7s，重复30次)，然后12000g，4℃离心10min，取上清液置冰上待测。

③ 血清 (浆)：可直接测定。若样本需要稀释，可用工作提取液进行适当稀释 (保证A_1测定大于1.0且ΔA_测定小于0.8)。

3. 实验步骤

① 酶标仪准备：预热30min以上，调节波长到340nm。

② 工作试剂的配制：临用前配制；按照试剂一：试剂二：试剂三：试剂四=1600μL：150 μL：150 μL：10 μL的比例混合均匀 (每14个反应孔的配制量)。

③ 检测体系配制：在96孔UV板中按照如下方式操作：

试剂	测定孔 (μL)	对照孔 (μL)
样本	10	10
工作试剂七	90	0
工作试剂六	0	90
/	混匀，室温 (25℃) 孵育30min	/
工作试剂	130	130
工作试剂五	20	20

注意：可在加入工作试剂前 (即室温孵育30 min后) 将工作试剂与工作试剂五按照每个反应需要130μL以及20μL的比例预混，然后每个反应孔中加入150μL混合后试剂。

4. 吸光度检测

迅速混匀，立即测定在340 nm 的吸光度值A_1测定和A_1对照以及在 2525℃下反应 5min 后的A_2测定和A_2对照。

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。

1. 数据处理计算ΔA_对照=A_1对照-A_2对照，ΔA_测定=A_1测定-A_2测定。 2. 样本GPX4酶活计算

① 按样本质量计算：

酶活单位定义：在25℃下，每g样品每分钟催化 1nmol NADPH 氧化定义为1个酶活单位。

GPX4 (U/g)=[(ΔA_测定-ΔA_对照)÷(ε×d)×V_反总×10⁹]÷(V_样÷V_样总×W)÷T=535.9×(ΔA_测定-ΔA_对照)÷W

② 按细胞或细菌数量计算：

酶活单位定义：在25℃下，每10⁶个细胞或细菌每分钟催化 1nmol NADPH 氧化定义为1个酶活单位。

GPX4 (U/10⁶)=[(ΔA_测定-ΔA_对照)÷(ε×d)×V_反总×10⁹]÷(N×V_样÷V_样总)÷T=535.9×(ΔA_测定-ΔA_对照)÷N

③ 按液体体积计算：

酶活单位定义：在25℃下，每mL液体每分钟催化 1nmol NADPH 氧化定义为1个酶活单位。

GPX4 (U/mL)=[(ΔA_测定-ΔA_对照)÷(ε×d)×V_反总×10⁹]÷V_样÷T=1071.8×(ΔA_测定-ΔA_对照)

④ 按蛋白浓度计算：

酶活单位定义：在25℃下，每mg蛋白每分钟催化 1nmol NADPH 氧化定义为1个酶活单位。

GPX4 (U/mg)=[(ΔA_测定-ΔA_对照)÷(ε×d)×V_反总×10⁹]÷(Cpr×V_样)÷T=1071.8×(ΔA_测定-ΔA_对照)÷Cpr

ε：NADPH摩尔消光系数，6.22×10³L/mol/cm；

d：96孔UV板光径，0.75cm；

V_反总：反应体系总体积，250μL=2.5×10⁻⁴ L；

10⁹：1 mol=1×10⁹ nmol；

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，10μL=1×10⁻²mL；

V_样总：提取液体积，0.5mL；

T：反应时间，5min；

N：细胞或细菌数量，以10⁶为单位 (例如细胞数量为5×10⁶，N=5)。

结果展示：

示例：取 0.121g 小鼠肾脏，匀浆上清稀释20倍后，按照测定步骤操作，用96孔板进行检测。

测得ΔA_测定=A_1测定-A_2测定=1.433-0.825=0.608， ΔA_对照=A_1对照-A_2对照=1.457-0.912=0.545。

按样本质量计算稀释 20 倍后的小鼠肾脏匀浆样本酶活为：GPX4 (U/g)=535.9×(ΔA_测定-ΔA_对照)÷W=279.02 U/g。

常见问题：

① 此试剂盒检测的结果与ELISA产品检测的GPX4含量的结果有什么差异？

答：此产品结果表示样本中GPX4酶的催化活性，与ELISA检测出的GPX4蛋白浓度结果表示的意义不同，可能会出现GPX4翻译后修饰导致蛋白浓度水平不变，但催化活性显著下降的现象。

② 检测得到的样本GPX4酶活过低或总GPX酶活过高怎么判断？

答：如果ΔA_测定-ΔA_对照小于0.005，表明样本中GPX4酶活过低，可将A_2测定和A_2对照延长至10min检测，并相应调整计算公式中的T为10，或适当加大样本量后，再重新测定；如果A_1测定小于1.0或ΔA_测定大于0.8，样本可用工作提取液进一步稀释或减少提取用样本量，再重新测定。

Glutathione peroxidase 4 (GPX4, https://ec.1.11.1.12) is the only monomeric seleno‑containing antioxidant enzyme in mammals that can directly reduce membrane‑bound lipid peroxides using GSH, thereby protecting the lipid bilayer from oxidative damage. With selenocysteine as its active center, GPX4 participates in vital physiological functions including cell membrane antioxidant defense, ferroptosis inhibition, and sperm maturation.

The detection principle of this kit is as follows: Glutathione peroxidase (GPX) catalyzes the oxidation of GSH by peroxides to generate GSSG. Glutathione reductase (GR) catalyzes the reduction of GSSG by NADPH to regenerate GSH. Meanwhile, NADPH is oxidized to NADP⁺. The GPX activity in samples can be calculated by measuring the rate of decrease in NADPH absorbance at 340 nm. By adding a GPX4 inhibitor to the reaction system, the total GPX activity and non‑specific enzyme activity excluding GPX4 can be determined. The difference between the two values represents GPX4 enzyme activity.

Contents & Storage:

Component No.	Component Name	Appearance	48T	96T	Storage
G1527167A	Extraction Buffer I	Liquid	30 mL	60 mL	-20℃.
G1527167B	Extraction Buffer II	Liquid	0.3 mL	0.6 mL	-20℃.
G1527167C	Reagent I	Liquid	15 mL	30 mL	-20℃.
G1527167D	Reagent II	Liquid	0.75 mL	1.5 mL	-20℃.
G1527167E	Reagent III	Liquid	0.75 mL	1.5 mL	-20℃.
G1527167F	Reagent IV	Liquid	50 μL	100 μL	-20℃.
G1527167G	Reagent V	Liquid	0.15 mL	0.3 mL	-20℃.
G1527167H	Reagent VI	Liquid	0.6 mL	1.2 mL	-20℃.
G1527167I	Reagent VII	Liquid	0.6 mL	1.2 mL	-20℃.

*Please check the quantity of each component before the experiment.
*Each component is supplied with an extra 10 % over the specified amount for preliminary experiments.

Self-supplied Instruments and Reagents:

Type of Self-supplied Resources	Resource Name	Remarks
Instruments	Microplate reader	Capable of measuring absorbance at 340 nm.
Consumables	96‑well microplate	UV‑plate
Reagents	PBS, Normal saline	For sample washing.
Others	Homogenizer (for tissue samples), incubator, ice box, low‑temperature centrifuge, adjustable pipettes and pipette tips.	Perform sample homogenization entirely on ice / under low‑temperature conditions.

Precautions:

1. Before official detection, it is recommended to carry out a preliminary experiment using 2‑3 samples with large expected differences.

2. For tissue, cell and other specimens, normalize test results among samples via protein‑concentration determination. The BCA Protein Quantification Kit (Aladdin Cat. No. B665595) is recommended.

3. The kit supports spectrophotometer detection. Adjust the dosage of prepared detection reagents proportionally to meet spectrophotometer‑based testing requirements.

4. Most biochemical assay reagents are irritant and biologically hazardous. For your safety and wellbeing, maintain complete biosafety protection in the whole experimental process. Put on a lab coat, respirator, gloves, hair‑net and other protective equipment. Perform experimental operations in a fume hood or biosafety cabinet.

5. This product is for scientific‑research use only and shall not be adopted for clinical diagnosis.

Experimental Procedure:

1. Reagent Preparation

Reagent Name	Reagent Preparation	Notes
Extraction Buffer I	Ready‑to‑use; equilibrate to room temperature before use.	Store at −20 °C.
Extraction Buffer II	Ready‑to‑use; equilibrate to room temperature before use.	Store at −20 °C.
Working Extraction Buffer	Prepare immediately before use: add 10 μL of Extraction Solution II into every 1 mL of Extraction Solution I.	Valid only on the day of preparation.
Reagent I	Ready‑to‑use; equilibrate to room temperature before use.	Store at −20 °C.
Reagent II	Ready‑to‑use; equilibrate to room temperature before use.	Store at −20 °C.
Reagent III	Ready‑to‑use; equilibrate to room temperature before use.	Store at −20 °C.
Reagent IV	Ready‑to‑use; equilibrate to room temperature before use.	Store at −20 °C.
Reagent V	Prepare immediately before assay: add 0.1 mL of Reagent V into every 0.9 mL of Reagent I.	Prepare prior to detection; valid within 4 hours.
Reagent VI	Prepare immediately before assay: add 0.2 mL of Reagent VI into every 0.8 mL of Reagent I.	Prepare prior to detection; valid within 4 hours.
Reagent VII	Prepare immediately before assay: add 0.2 mL of Reagent VII into every 0.8 mL of Reagent I.	Prepare prior to detection; valid within 4 hours.

2. Sample Preparation

Note: Fresh samples are recommended. Enzyme‑activity results may be reduced for frozen samples. All sample‑handling procedures must be performed on ice, and enzyme activity shall be assayed on the same day.

① Animal tissues: Rinse tissues with cold PBS to remove blood as thoroughly as possible. Blot surface moisture and weigh the tissue. Weigh 0.1 g tissue sample and add 0.5 mL Working Extraction Buffer, then rapidly homogenize on ice. Centrifuge the homogenate at 12 000 g for 10 min at 4 °C. Transfer the supernatant to ice for subsequent assay. Tissue samples are recommended to be diluted 20‑fold with Working Extraction Buffer before detection; liver samples are recommended to be diluted 40‑fold.

② Cell (bacterial) samples: Collect 5 × 10⁶ cells (bacteria) into a centrifuge tube, add 0.5 mL Working Extraction Buffer. Lyse cells (bacteria) by sonication on ice for 5 min (power: 20 % or 200 W; sonicate 3 s, interval 7 s; repeat for 30 cycles). Centrifuge at 12 000 g for 10 min at 4 °C. Transfer the supernatant to ice for subsequent assay.

③ Serum (plasma): Can be assayed directly. If sample dilution is required, dilute appropriately with Working Extraction Buffer (ensure that A_1‑sample > 1.0 and ΔA_sample  < 0.8).

3. Experimental Procedures

① Microplate reader preparation: Warm‑up for no less than 30 min and set the wavelength to 340 nm.

② Working‑reagent preparation: Prepare immediately before use. Thoroughly mix Reagent I : Reagent II : Reagent III : Reagent IV at a volume ratio of 1600 μL : 150 μL : 150 μL : 10 μL (formulation volume for 14 reaction wells).

③ Preparation of detection system: Perform operations in a 96‑well UV‑plate as shown below:

Reagent	Sample Well (μL)	Blank Well (μL)
Sample	10	10
Working Reagent VII	90	0
Working Reagent VI	0	90
/	Mix well and incubate at room temperature (25 °C) for 30 min.	/
Working Reagent	130	130
Working Reagent V	20	20

Note: Prior to adding to wells (after the 30‑min room‑temperature incubation), Working Reagent and Working Reagent V may be pre‑mixed at the ratio of 130 μL and 20 μL per reaction, and 150 μL of the mixture is added to each well.

4. Absorbance Measurement

Mix rapidly. Immediately measure absorbance values A_1‑sample and A_1‑blank at 340 nm, as well as A_2‑sample and A_2‑blank after 5 min of reaction at 25 °C.

Result Calculation:

Both the derived formula and simplified formula provided below are mathematically equivalent.

1. Data processing Calculate ΔA_blank = A_1‑blank − A_2‑blank, ΔA_sample = A_1‑sample − A_2‑sample.

2. Calculation of GPX4 enzyme activity in samples

① Calculation based on sample weight Definition of enzyme‑activity unit:

One unit (U) is defined as the amount of enzyme that catalyzes the oxidation of 1 nmol NADPH per minute per gram of sample at 25 °C.

GPX4 (U/g) = [(ΔA_sample − ΔA_blank) ÷ (ε × d) × V_{total reaction} × 10⁹] ÷ (V_sample ÷ V_{total‑sample} × W) ÷ T= 535.9 × (ΔA_sample − ΔA_blank) ÷ W

② Calculation based on cell or bacterial count Definition of enzyme‑activity unit:

One unit (U) is defined as the amount of enzyme that catalyzes the oxidation of 1 nmol NADPH per minute per 10⁶ cells or bacteria at 25 °C.

GPX4 (U/10⁶ cells) = [(ΔA_sample − ΔA_blank) ÷ (ε × d) × V_{total reaction} × 10⁹] ÷ (N × V_sample ÷ V_{total‑sample}) ÷ T= 535.9 × (ΔA_sample − ΔA_blank) ÷ N

③ Calculation based on liquid volume Definition of enzyme‑activity unit:

One unit (U) is defined as the amount of enzyme that catalyzes the oxidation of 1 nmol NADPH per minute per milliliter of liquid sample at 25 °C.

GPX4 (U/mL) = [(ΔA_sample − ΔA_blank) ÷ (ε × d) × V_{total reaction} × 10⁹] ÷ V_sample ÷ T= 1071.8 × (ΔA_sample − ΔA_blank)

④ Calculation based on protein concentration Definition of enzyme‑activity unit:

One unit (U) is defined as the amount of enzyme that catalyzes the oxidation of 1 nmol NADPH per minute per milligram of protein at 25 °C.

GPX4 (U/mg protein) = [(ΔA_sample − ΔA_blank) ÷ (ε × d) × V_{total reaction} × 10⁹] ÷ (Cpr × V_sample) ÷ T= 1071.8 × (ΔA_sample − ΔA_blank) ÷ Cpr

ε: Molar extinction coefficient of NADPH, 6.22 × 10³ L/(mol·cm);

d: Optical path length of 96‑well UV‑plate, 0.75 cm;

V_{total reaction}: Total volume of reaction system, 250 μL = 2.5 × 10⁻⁴ L;

10⁹: Conversion factor, 1 mol = 1 × 10⁹ nmol;

Cpr: Protein concentration of supernatant (mg/mL);

W: Sample weight (g);

V_sample: Volume of supernatant added to reaction system, 10 μL = 1 × 10⁻² mL;

V_{total‑sample}: Extraction solution volume, 0.5 mL;

T: Reaction time, 5 min;

N: Number of cells or bacteria in units of 10⁶ (e.g., if cell count equals 5 × 10⁶, N = 5).

Result Presentation:

Example: 0.121 g of mouse kidney tissue was collected. The homogenate supernatant was diluted 20‑fold and assayed following the detection procedure using a 96‑well plate.

The measured values were ΔA_sample = A_1‑sample − A_2‑sample = 1.433 − 0.825 = 0.608, and ΔA_blank = A_1‑blank − A_2‑blank = 1.457 − 0.912 = 0.545.

Calculated by sample weight, the enzyme activity of the 20‑fold‑diluted mouse‑kidney homogenate sample was:

GPX4 (U/g) = 535.9 × (ΔA_sample − ΔA_blank) ÷ W = 279.02 U/g.

Frequently‑asked Questions:

① What is the difference between results obtained with this kit and GPX4 levels measured by ELISA? Answer: Results from this kit reflect the catalytic activity of GPX4 in samples, which differs in biological meaning from GPX4 protein concentrations measured by ELISA. Post‑translational modification of GPX4 may occur, whereby protein abundance remains unchanged while catalytic activity decreases markedly.

② How to troubleshoot excessively low sample GPX4 activity or abnormally high total GPX activity?

Answer: If ΔA_sample − ΔA_blank is less than 0.005, GPX4 activity in the sample is too low. Extend the reaction time for acquiring A_2‑sample and A_2‑blank to 10 min, adjust T in the formula to 10 accordingly, or increase sample loading appropriately before re‑measurement. If A_1‑sample is lower than 1.0 or ΔA_sample is greater than 0.8, further dilute the sample with Working Extraction Solution or reduce the starting sample amount for homogenization before re‑measurement.

规格

规格或纯度

BioReagent

英文名称

Glutathione Peroxidase 4 (GPX4) Activity Assay Kit (NADPH Rate, UV Micro Method)

应用

酶活性检测

样品类型

动植物组织, 细胞, 细菌, 血浆, 血清

检测方法

分光光度法

检测仪器

酶标仪

检测波长

400nm

敏感性

Light-sensitive

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

Store at -20℃ long term (12 months).

组分与储存

组分货号	组分名称	物理外观	48T	96T	储存
G1527167A	提取液一	液体	30 mL	60 mL	-20℃.
G1527167B	提取液二	液体	0.3 mL	0.6 mL	-20℃.
G1527167C	试剂一	液体	15 mL	30 mL	-20℃.
G1527167D	试剂二	液体	0.75 mL	1.5 mL	-20℃.
G1527167E	试剂三	液体	0.75 mL	1.5 mL	-20℃.
G1527167F	试剂四	液体	50 μL	100 μL	-20℃.
G1527167G	试剂五	液体	0.15 mL	0.3 mL	-20℃.
G1527167H	试剂六	液体	0.6 mL	1.2 mL	-20℃.
G1527167I	试剂七	液体	0.6 mL	1.2 mL	-20℃.

*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%的量用于进行预实验。

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🔬 规格说明书

该级别的完整质量属性和验收标准。

查看规格说明 →

高级数据

质检证书（CoA,COO,BSE/TSE 和分析图谱)

C of A & Other Certificates(BSE/TSE, COO):

输入批号以搜索分析图谱:

溶液计算器

摩尔浓度计算器

计算溶液所需的质量、体积或浓度。

质量

浓度

体积

分子量

g/mol

稀释计算器

计算配制储备溶液所需的稀释度。

浓度1（C1）

体积 1 (V1)

浓度 2 (C2)

体积 2 (V2)

复溶计算器

复溶计算器允许您快速计算试剂的体积，以复溶小瓶。只需输入试剂的质量和目标浓度，计算器将确定其余部分。

体积（添加到小瓶中）

质量（小瓶）

所需的复溶浓度

需要帮助选择级别？

我们的级别选择指南涵盖了目录中所有的变体的纯度、稳定剂状态和应用适用性。

查看 BioReagent 级别指南 →

大包装与定制包装 需要更大包装或定制规格？请联系我们的团队获取专属报价。

谷胱甘肽过氧化物酶4活性检测试剂盒 (NADPH速率, 紫外微量法)