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硫氧还蛋白还原酶（ThioredoxinReductase, TrxR）是一种NADPH依赖的二聚体硒酶，包含FAD结构域，属于吡啶核苷酸-二硫化物氧化还原酶家族成员，与硫氧还蛋白以及NADPH共同构成了硫氧还蛋白系统。TrxR 与GR活性类似，催化GSSG还原生成GSH，是谷胱甘肽氧化还原循环关键酶之一。
检测原理：TrxR催化NADPH还原DTNB生成TNB和NADP⁺，TNB在412nm有特征吸收峰，但还原型谷胱甘肽与DTNB同样能反应生成TNB，利用2-乙烯吡啶抑制样本中原有的还原型谷胱甘肽，通过测定412nm波长处TNB的增加速率，即可计算TrxR活性。
适用样本：动物组织、细胞、细菌、血清（浆）
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
酶标仪（能测412nm处的吸光度）
96 孔酶标板（普通透明板）
去离子水/PBS（样本稀释用/样本清洗用）
匀浆器（组织样本）、恒温水浴锅、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）
操作步骤
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 |
| Working Chromogen | 临用前配制，Chromogen加入3mL去离子水溶解 | 用不完的试剂4℃避光保存可稳定2周；Chromogen有一定的刺激性，建议在使用过程中做好个人防护 |
| Working TrxR Cofactor | 临用前配制，TrxR Cofactor加入5mL去离子水溶解 | 用不完的试剂-20℃避光分装保存2周，避免反复冻融 |
| Inhibitor | 即用型，使用前平衡到室温 | -20℃避光保存；Inhibitor有一定的刺激性，建议在使用过程中做好个人防护 |
2、样本制备
注意：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。推荐使用新鲜样本，如果不立即进行实验，样本可在-80℃保存一个月。细胞中TrxR活性测定时，细胞数目须在3-5×10⁶之间，提取细胞中TrxR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。
2.1 动物组织
称取约0.1g组织，加入1mL 预冷的Assay Buffer，冰浴匀浆，10000g，4℃离心10min，取上清液，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 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），4℃，10000g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
注意：哺乳动物组织及血液制品TrxR活力测定时，一般须用去离子水稀释5倍左右；测定过程操作须迅速。由于Assay Buffer中含有一定浓度的蛋白（约0.1mg/mL），所以在测定样品蛋白浓度时需要减去Assay Buffer本身的蛋白含量。
3.1 酶标仪准备
预热30min以上，调节波长到412nm。Assay Buffer置于25℃（一般物种）或者37℃（哺乳动物）孵育30min。
3.2 检测体系配制
空白孔只需做1个即可。在96孔板中按照如下方式操作：
|
3.3 吸光度检测
迅速混匀，立即测定412nm处第10s时吸光度为A1，置于25℃（一般物种）或者37℃（哺乳动物）孵育5min，记录第5min10s时吸光值为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=A2-A1，ΔA测定=A2测定-A1测定，ΔA空白=A2空白-A1空白，ΔΔA=ΔA测定-ΔA空白
4.2 样本TrxR活性计算
(1) 按样本蛋白浓度计算
活性单位（U）定义：在25℃或者37℃中，每毫克蛋白每分钟催化1μmol DTNB还原。
TrxR (U/mg prot)=ΔΔA÷(ε×d)×V反总÷(Cpr×V样)÷T=0.294×ΔΔA÷Cpr
(2) 按样本鲜重计算
活性单位（U）定义：在25℃或者37℃中，每克样品每分钟催化1μmol DTNB还原。
TrxR (U/g)=ΔΔA÷(ε×d)×V反总÷(V样÷V样总×W)÷T=0.294×ΔΔA÷W
(3) 按细胞或细菌密度计算
活性单位（U）定义：在25℃或者37℃中，每10⁴个细胞或细菌每分钟催化1μmol DTNB还原。 TrxR (U/10⁴)=ΔΔA÷(ε×d)×V反总÷(N×V样÷V样总)÷T=0.294×ΔΔA÷N
(4) 按样本体积计算
活性单位（U）定义：在25℃或者37℃中，每毫升液体每分钟催化1μmol DTNB还原。
TrxR (U/mL)=ΔΔA÷(ε×d)×V反总÷V样÷T=0.294×ΔΔA
参数说明：
ε：产物微摩尔消光系数，0.0136L/μmol/cm；
d：96孔板光径，0.5cm；
V反总：反应体系总体积，200μL=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：提取液体积，1mL；
T：反应时间，5min；
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
示例-1：取0.1 g小鼠肝脏，按照测定步骤操作，用96孔板进行检测。测得ΔA空白=A2-A1=0，ΔA测定=A2测定-A1测定=1.772-1.109=0.663。按样本质量计算得：TrxR (U/g)=0.294×ΔΔA÷W=1.949 U/g。
示例-2: 取0.1 g小鼠肾脏，按照测定步骤操作，用96孔板进行检测。测得ΔA空白=A2-A1=0，ΔA测定=A2测定-A1测定=1.494-0.954=0.540。按样本质量计算得：TrxR (U/g)=0.294×ΔΔA÷W=1.588 U/g。
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，使得吸光值在5min内呈线性变化。
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。
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。
Thioredoxin Reductase (TrxR) is an NADPH-dependent dimeric selenoenzyme containing an FAD domain, belonging to the pyridine nucleotide-disulfide oxidoreductase family. Together with thioredoxin and NADPH, it constitutes the thioredoxin system. TrxR activity is similar to that of GR (glutathione reductase), catalyzing the reduction of GSSG to GSH, making it one of the key enzymes in the glutathione redox cycle.
Detection Principle
TrxR catalyzes the reduction of DTNB by NADPH to generate TNB and NADP⁺. TNB has a characteristic absorption peak at 412 nm. However, reduced glutathione (GSH) can also react with DTNB to produce TNB. The original reduced glutathione in the sample is inhibited using 2-vinylpyridine. By measuring the rate of increase in TNB at 412 nm, TrxR activity can be calculated.
Applicable Samples: Animal tissues, cells, bacteria, serum (plasma)
Please check the quantity of each component before the experiment.
An additional 10% of each component is provided based on the specification for preparing standard curves or preliminary experiments.
Reagents, consumables and Equipments not provided
Microplate reader (capable of measuring absorbance at 412 nm)
96-well microplate (standard transparent plate)
Deionized water/PBS (for sample dilution/washing)
Homogenizer (for tissue samples), Constant temperature water bath, Low-temperature centrifuge, Adjustable pipettes and tips (using multichannel pipettes is recommended for efficiency when handling large numbers of samples)
Procedure
1. Reagent Preparation
| Reagent Name | Preparation | Notes |
| Assay Buffer | Ready-to-use, equilibrate to room temperature before use. | Store at 4°C. |
| Working Chromogen | Prepare immediately before use. Dissolve Chromogen in 3 mL deionized water. | Unused reagent can be stored at 4°C protected from light for up to 2 weeks. Chromogen is somewhat irritating; personal protection is recommended. |
| Working TrxR Cofactor | Prepare immediately before use. Dissolve TrxR Cofactor in 5 mL deionized water. | Unused reagent can be aliquoted and stored at -20°C protected from light for 2 weeks. Avoid repeated freeze-thaw cycles. |
| Inhibitor | Ready-to-use, equilibrate to room temperature before use. | Store at -20°C protected from light. Inhibitor is somewhat irritating; personal protection is recommended. |
2. Sample Preparation
*Note: All sample processing should be performed on ice. Enzyme activity must be measured on the same day. Fresh samples are recommended. If not used immediately, samples can be stored at -80°C for one month. For measuring TrxR activity in cells, the cell number should be between 3-5×10⁶. When extracting TrxR from cells, homogenize or sonicate in Assay Buffer; do not use cell lysis buffer.*
2.1 Animal Tissue
Weigh approximately 0.1 g of tissue, 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 keep it on ice for assay.
2.2 Cells or Bacteria
Collect 5×10⁶ cells or bacteria, wash with pre-cooled PBS, and centrifuge at 800 g for 2 minutes. Discard the supernatant. Add 1 mL of Assay Buffer and disrupt by sonication on ice. Sonicate for 5 minutes (power 20% or 200 W, pulse 3 seconds, interval 7 seconds, repeat 30 times). Centrifuge at 4°C, 10,000 g for 10 minutes. Collect the supernatant and keep it on ice for assay.
2.3 Serum (Plasma)
Assay directly.
3. Experimental Steps
*Note: For mammalian tissues and blood products, TrxR activity usually needs to be diluted about 5-fold with deionized water. The assay procedure should be performed swiftly. Since the Assay Buffer contains a certain concentration of protein (~0.1 mg/mL), this background protein content must be subtracted when determining sample protein concentration.*
3.1 Microplate Reader Preparation
Preheat for at least 30 minutes and set the wavelength to 412 nm. Incubate the Assay Buffer at 25°C (for general species) or 37°C (for mammals) for 30 minutes.
3.2 Assay System Setup
Only one blank well is needed. Perform the following steps in a 96-well plate:
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3.3 Absorbance Measurement
Mix rapidly immediately after adding all reagents. Measure the absorbance at 412 nm at the 10-second mark, recorded as A₁. Incubate the plate at 25°C (general species) or 37°C (mammals) for 5 minutes. Measure the absorbance at the 5-minute 10-second mark, recorded as A₂. Record values for the test well as A<sub>Test</sub> and for the blank well as A<sub>Blank</sub>.
4. Calculation of Results
Both the derived and simplified calculation formulas provided below are completely equivalent.
4.1 Data Processing
Calculate ΔA = A₂ - A₁, ΔATest = A₂Test - A₁Test, ΔABlank = A₂Blank - A₁Blank. Then calculate ΔΔA = ΔATest - ΔABlank.
4.2 Sample TrxR Activity Calculation
(1) Based on sample protein concentration
Unit definition: One unit (U) is the amount of enzyme that catalyzes the reduction of 1 µmol of DTNB per minute per mg of protein at 25°C or 37°C.
TrxR (U/mg prot) = ΔΔA ÷ (ε × d) × VTotal reaction ÷ (Cpr × VSample) ÷ T = 0.294 × ΔΔA ÷ Cpr
(2) Based on sample fresh weight
Unit definition: One unit (U) is the amount of enzyme that catalyzes the reduction of 1 µmol of DTNB per minute per gram of sample at 25°C or 37°C.
TrxR (U/g) = ΔΔA ÷ (ε × d) × VTotal reaction ÷ (VSample ÷ VTotal extract × W) ÷ T = 0.294 × ΔΔA ÷ W
(3) Based on cell or bacterial density
Unit definition: One unit (U) is the amount of enzyme that catalyzes the reduction of 1 µmol of DTNB per minute per 10⁴ cells or bacteria at 25°C or 37°C.
TrxR (U/10⁴) = ΔΔA ÷ (ε × d) × VTotal reaction ÷ (N × VSample ÷ VTotal extract) ÷ T = 0.294 × ΔΔA ÷ N
(4) Based on sample volume
Unit definition: One unit (U) is the amount of enzyme that catalyzes the reduction of 1 µmol of DTNB per minute per milliliter of liquid at 25°C or 37°C.
TrxR (U/mL) = ΔΔA ÷ (ε × d) × VTotal reaction ÷ VSample ÷ T = 0.294 × ΔΔA
Parameter Description:
ε: Molar extinction coefficient of the product, 0.0136 L/µmol/cm
d: Light path of the 96-well plate, 0.5 cm
V<sub>Total reaction</sub>: Total reaction volume, 200 µL = 2×10⁻⁴ L
Cpr: Protein concentration of the supernatant, mg/mL
W: Sample mass, g
V<sub>Sample</sub>: Volume of supernatant added to the reaction system, 20 µL = 0.02 mL
VTotal extract: Volume of extraction buffer, 1 mL
T: Reaction time, 5 min
N: Number of cells or bacteria, in units of 10⁴ (e.g., for 5×10⁶ cells, N=500).
5. Example Results
Example 1: 0.1 g of mouse liver was processed and assayed in a 96-well plate. ΔABlank = A₂ - A₁ = 0, ΔATest = A₂Test - A₁Test = 1.772 - 1.109 = 0.663. TrxR activity calculated based on sample weight: TrxR (U/g) = 0.294 × 0.663 ÷ 0.1 = 1.949 U/g.
Example 2: 0.1 g of mouse kidney was processed and assayed in a 96-well plate. ΔABlank = A₂ - A₁ = 0, ΔATest = A₂Test - A₁Test = 1.494 - 0.954 = 0.540. TrxR activity calculated based on sample weight: TrxR (U/g) = 0.294 × 0.540 ÷ 0.1 = 1.588 U/g.
Notes
Before formal testing, it is recommended to perform a preliminary experiment with 2-3 samples expected to show significant differences, ensuring the absorbance change is linear within 5 minutes.
For tissue and cell samples, results can be normalized by measuring protein concentration. We recommend using Aladdin B665595 BCA Protein Quantification Kit or R1491648 Ready-to-use BCA Protein Quantification Kit.
This kit is compatible with spectrophotometer detection. Adjust the reagent preparation amounts proportionally according to the spectrophotometer's requirements.
Biochemical reagents are generally irritating and may have biological toxicity. For your safety and health, maintain biosafety precautions throughout the experiment: wear a lab coat, mask, gloves, head cover, etc., and perform experiments in a fume hood or biosafety cabinet.
This product is for scientific research use only and is not intended for clinical diagnosis.
FAQ
Q: What should I do if the sample ΔΔA obtained is too high or too low?
A: If ΔΔA is less than 0.02, you can appropriately increase the sample amount. If ΔΔA is greater than 1.0, further dilute the sample with Assay Buffer, or reduce the amount of sample used for extraction, and repeat the assay.
| S1507969 | Component | 48T | 96T | Storage |
| S1507969A | Assay Buffer | 70 mL | 70 mL×2 | 2-8℃ |
| S1507969B | Chromogen | 1 EA | 1 EA | 2-8℃. Store in the dark. |
| S1507969C | TrxR Cofactor | 1 EA | 1 EA | -20℃. Store in the dark. |
| S1507969D | Inhibitor | 60 μL | 120 μL | -20℃. Store in the dark. |
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| 批号(Lot Number) | 证书类型 | 货号 |
|---|---|---|
 J2608227 |
分析证书 | S1507969 |
| ZJ25F1230488 |
分析证书 | S1507969 |
| ZJ25F1230489 |
分析证书 | S1507969 |
| ZJ25F1230490 |
分析证书 | S1507969 |
| ZJ25F1230491 |
分析证书 | S1507969 |
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