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酮酸激酶（Pyruvate Kinase，PK，EC2.7.1.40）广泛存在于动物、植物、微生物和培养的细胞中，催化糖酵解过程中的最后一步反应，是糖酵解过程中的主要限速酶之一，也是产生ATP的关键酶之一，因此测定PK活性具有重要意义。
检测原理：PK催化磷酸烯醇式丙酮酸和ADP生成ATP和丙酮酸，乳酸脱氢酶进一步催化NADH和丙酮酸生成乳酸和NAD⁺，测定NADH在340nm处吸光度值的变化即可计算样本中PK的活性。
适用样本：动植物组织、细胞、细菌、血清（浆）
| A1501205 | Component | 48T | 96T | Storage |
| A1501205A | Extraction Buffer | 60 mL | 60 mL×2 | 2-8℃ |
| A1501205B | Assay Buffer | 12 mL | 24 mL | 2-8℃ |
| A1501205C | Substrate Mix | 1EA | 1EA | -20℃. Store in the dark. |
| A1501205D | LDH | 10.2 μL | 20.4 μL | 2-8℃ |
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340nm处的吸光度 |
| 耗材 | 96孔UV板 | 紫外透明板 |
| 试剂 | PBS (pH7.4) /去离子水 | 清洗细胞（菌）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 |
| Extraction Buffer | 即用型；使用前平衡到室温 | 4℃保存 |
| Assay Buffer | 即用型；使用前平衡到室温 | 4℃保存 |
| Substrate Mix Working Reagent | 临用前配制； 48T加入10.2mL Assay Buffer和0.6mL 去离子水； 96T加入20.4mL Assay Buffer 和1.2mL 去离子水； 充分溶解 | 分装后-20℃保存6个月，避免反复冻融。 使用前置于25℃（一般物种）或者 37℃（哺乳动物）孵育5min。 |
| LDH Working Reagent | 临用前配制； 48T加入0.6mL 去离子水； 96T加入1.2mL 去离子水； 充分混匀 | 置冰上待用；稀释后4℃保存1个月 |
2、样本制备
注意：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。
2.1 动植物组织样本：称取约0.1g样本，加入1mL Extraction Buffer，冰浴匀浆，8000g，4℃离心10min，取上清液置冰上待测。
2.2 细胞（菌）：收集5×10⁶ 个细胞或细菌到离心管内，用冷PBS清洗细胞或细菌，离心后弃上清，加入1mL Extraction Buffer，冰浴超声破碎5min（功率20%或200W，超声3s，间隔7s，重复30次），然后8000g，4℃离心10min，取上清液置冰上待测。
2.3 血清（浆）和其他液体样本：直接检测。
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 检测体系配制：在96孔UV板中依次加入10μL样本，10μL LDH Working Reagent和180μL Substrate Mix Working Reagent，迅速混匀。
3.3 吸光度检测：混匀后立即测定340nm处第20s时的吸光度值和第2min20s时的吸光度值，分别记为A1和A2。
4、结果计算
4.1 数据处理
计算ΔA=A1-A2。
4.2 样本PK活性计算
(1) 按样本质量计算
酶活单位的定义：每g组织在反应体系中每分钟消耗1nmol的NADH定义为一个酶活力单位。
K (U/g)＝[ΔA×V反总÷(ε×d)× 10⁹ ]÷(V样÷V样总×W)÷T=3215×ΔA÷W
(2) 按细胞（菌）数量计算
酶活单位的定义：每10⁴个细菌或细胞在反应体系中每分钟消耗1nmol的NADH定义为一个酶活力单位。
PK (U/ 10⁴)＝[ΔA×V反总 ÷(ε×d)×10⁹]÷(V样÷V样总×500)÷T=3215×ΔA÷500=6.431×ΔA
(3) 按液体体积计算
酶活单位的定义：每mL血清（浆）在反应体系中每分钟消耗1nmol的NADH定义为一个酶活力单位。
PK (U/mL)=[ΔA×V反总÷(ε×d)×10⁹]÷V样÷T=3215×ΔA
(4) 按蛋白浓度计算
酶活单位的定义：每mg蛋白在反应体系中每分钟消耗1nmol的NADH定义为一个酶活力单位。
PK (U/mg prot)＝[ΔA×V反总÷(ε×d)×10⁹]÷(Cpr×V样)÷T=3215×ΔA÷Cpr
参数说明
V反总：反应体系总体积，2×10⁻⁴ L；
ε：NADH摩尔消光系数，6.22×10³ L/mol/cm；
d：96孔板光径，0.5cm；
10⁹：1 mol=1× 10⁹ nmol；
V样：加入样本体积，0.01mL；
V样总：加入Extraction Buffer体积，1mL；
T：反应时间，2min；
Cpr：样本蛋白质浓度，mg/mL；
W：样本质量，g；
500：细胞或细菌数量，5×10⁶，以10⁴为单位。
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、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。
3、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。
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ΔA过高或者过低怎么办？
A：如果样本的ΔA＞1.0，则说明样本PK活性较高，需用Extraction Buffer适当稀释或减少提取用样本量，再重新进行测定。如果样本的ΔA＜0.005，可延长反应时间到5min或10min或者适当加大样本量，再重新进行测定。
Q：多个样本同时检测时会不会影响检测结果？
A：本项目适当延长时间3-5min不会影响检测结果，多样本检测时建议用排枪操作。
Pyruvate Kinase (PK, EC 2.7.1.40) is widely present in animals, plants, microorganisms, and cultured cells. It catalyzes the final step of glycolysis and is one of the key rate-limiting enzymes in glycolysis, as well as a crucial enzyme for ATP production. Therefore, determining PK activity is of significant importance.
Detection Principle: PK catalyzes the conversion of phosphoenolpyruvate and ADP to ATP and pyruvate. Lactate dehydrogenase (LDH) further catalyzes the reaction of NADH and pyruvate to produce lactate and NAD⁺. The change in NADH absorbance at 340 nm is measured to calculate the PK activity in the sample.
Applicable Samples: Animal/plant tissues, cells, bacteria, serum (plasma)
| A1501205 | Component | 48T | 96T | Storage |
| A1501205A | Extraction Buffer | 60 mL | 60 mL×2 | 2-8℃ |
| A1501205B | Assay Buffer | 12 mL | 24 mL | 2-8℃ |
| A1501205C | Substrate Mix | 1EA | 1EA | -20℃. Store in the dark. |
| A1501205D | LDH | 10.2 μL | 20.4 μL | 2-8℃ |
Please check the quantity of each component before the experiment.
An additional 10% of each component is provided beyond the specified volume for standard curve preparation or preliminary experiments.
User-Provided Instruments and Reagents
| Type | Name | Notes |
| Instrument | Microplate Reader | Capable of measuring absorbance at 340 nm. |
| Consumables | 96-well UV Plate | UV-transparent plate. |
| Reagents | PBS (pH 7.4) / Deionized Water | For washing cells/bacteria / Reagent preparation. |
| Others | Homogenizer (for tissue samples), incubator, ice bucket, low-temperature centrifuge, adjustable pipettes and tips | Using a multichannel pipette for large-scale detection can improve efficiency. |
Experimental Procedure
1. Reagent Preparation
| Reagent Name | Reagent Preparation | Precautions |
| Extraction Buffer | Ready-to-use; equilibrate to room temperature before use. | Store at 4°C. |
| Assay Buffer | Ready-to-use; equilibrate to room temperature before use. | Store at 4°C. |
| Substrate Mix Working Reagent | Prepare before use: For 48T: Add 10.2 mL Assay Buffer and 0.6 mL deionized water to the vial. For 96T: Add 20.4 mL Assay Buffer and 1.2 mL deionized water to the vial. Dissolve thoroughly. | After preparation, aliquot and store at -20°C for up to 6 months. Avoid repeated freeze-thaw cycles. Before use, incubate at 25°C (for general species) or 37°C (for mammals) for 5 min. |
| LDH Working Reagent | Prepare before use: For 48T: Add 0.6 mL deionized water to the LDH vial. For 96T: Add 1.2 mL deionized water to the LDH vial. Mix thoroughly. | Keep on ice after preparation. The diluted reagent can be stored at 4°C for 1 month. |
2. Sample Preparation
Note: Fresh samples are recommended. If not used immediately, samples can be stored at -80°C for up to 1 month.
2.1 Animal/Plant Tissues: Weigh approximately 0.1 g of tissue, add 1 mL of Extraction Buffer, and homogenize on ice. Centrifuge at 8000 g, 4°C for 10 minutes. Collect the supernatant and keep on ice for detection.
2.2 Cells/Bacteria: Collect 5×10⁶ cells or bacteria into a centrifuge tube. Wash with cold PBS, centrifuge, and discard the supernatant. Add 1 mL of Extraction Buffer. Disrupt by ultrasonic homogenization on ice (power 20% or 200 W, ultrasonicate for 3 s, interval 7 s, repeat 30 times). Centrifuge at 8000 g, 4°C for 10 minutes. Collect the supernatant and keep on ice for detection.
2.3 Serum (Plasma) and other liquid samples: Detect directly.
3. Assay Steps
3.1 Microplate Reader Preparation: Preheat for at least 30 minutes. Set the wavelength to 340 nm.
3.2 Assay System Setup: In a 96-well UV plate, add sequentially: 10 µL of sample, 10 µL of LDH Working Reagent, and 180 µL of Substrate Mix Working Reagent. Mix rapidly immediately after addition.
3.3 Absorbance Measurement: Immediately after mixing, measure the absorbance at 340 nm at 20 seconds (A1) and then at 2 minutes and 20 seconds (A2). Calculate ΔA = A1 - A2.
4. Result Calculation
4.1 Data Processing
Calculate ΔA = A1 - A2.
4.2 Sample PK Activity Calculation
(1) Based on sample mass:
Unit Definition: One unit of enzyme activity is defined as the consumption of 1 nmol NADH per minute per gram of tissue in the reaction system.
Formula:
PK (U/g) = [ΔA×Vtotal reaction÷ (ε × d) × 10⁹] ÷ (Vsample ÷ Vtotal extract×W) ÷ T = 3215 × ΔA ÷ W
(2) Based on cell/bacterial count:
Unit Definition: One unit of enzyme activity is defined as the consumption of 1 nmol NADH per minute per 10⁴ bacteria or cells in the reaction system.
Formula:
PK (U/10⁴) = [ΔA × Vtotal reaction ÷ (ε × d) × 10⁹] ÷ (Vsample ÷ Vtotal extract; × 500) ÷ T = 3215 × ΔA ÷ 500 = 6.431 × ΔA
(3) Based on liquid volume:
Unit Definition: One unit of enzyme activity is defined as the consumption of 1 nmol NADH per minute per mL of serum (plasma) in the reaction system.
Formula:
PK (U/mL) = [ΔA × Vtotal reaction; ÷ (ε × d) × 10⁹] ÷ Vsample ÷ T = 3215 × ΔA
(4) Based on protein concentration:
Unit Definition: One unit of enzyme activity is defined as the consumption of 1 nmol NADH per minute per mg of protein in the reaction system.
Formula:
PK (U/mg prot) = [ΔA × Vtotal reaction; ÷ (ε × d) × 10⁹] ÷ (Cpr × Vsample ) ÷ T = 3215 × ΔA ÷ Cpr
Parameter Description:
Vtotal reaction; : Total reaction volume, 2 × 10⁻⁴ L
ε: Molar extinction coefficient of NADH, 6.22 × 10³ L/mol/cm
d: Light path of the 96-well plate, 0.5 cm
10⁹: Conversion factor (1 mol = 1 × 10⁹ nmol)
Vsample : Volume of sample added, 0.01 mL
Vtotal extract; : Volume of Extraction Buffer added, 1 mL
T: Reaction time, 2 min
Cpr: Sample protein concentration, mg/mL
W: Sample mass, g
500: Cell or bacterial count (5 × 10⁶), converted to units of 1
Precautions
1. It is recommended to perform preliminary experiments using 2-3 samples expected to have significant differences before formal testing.
2. This kit is compatible with spectrophotometer detection. Adjust the preparation volume of detection reagents proportionally according to the spectrophotometer's requirements.
3. For tissue and cell samples, results can be normalized by measuring the protein concentration. Aladdin BCA Protein Quantification Kit (B665595) or Ready-to-Use BCA Protein Quantification Kit (R1491648) are recommended.
4. Biochemical reagents are generally irritating and biologically toxic. For your safety and health, please implement appropriate biosafety precautions throughout the experiment. Wear personal protective equipment such as lab coats, masks, gloves, and hair caps. Perform experiments in a fume hood or biosafety cabinet.
5. This product is for scientific research use only. Not intended for clinical diagnosis.
Frequently Asked Questions
Q: What should I do if the sample ΔA is too high or too low?
A: If the sample ΔA is > 1.0, the PK activity in the sample is too high. Dilute the sample appropriately with Extraction Buffer or reduce the amount of sample used for extraction, and then re-assay. If the sample ΔA is < 0.005, extend the reaction time to 5 or 10 minutes, or appropriately increase the sample amount, and then re-assay.
Q: Will testing multiple samples simultaneously affect the results?
A: Appropriately extending the time by 3-5 minutes for this assay will not affect the results. When testing multiple samples, it is recommended to use a multi-channel pipette for operation.
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| 批号(Lot Number) | 证书类型 | 货号 |
|---|---|---|
 ZJ25F1028975 |
分析证书 | A1501205 |
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