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磷酸果糖激酶（PFK，EC2.7.1.11）广泛存在于动物、植物、微生物和培养细胞中，负责将果糖-6-磷酸和ATP转化为果糖-1,6二磷酸和ADP，是糖酵解过程的关键调节酶之一。
检测原理：PFK催化果糖-6-磷酸和ATP生成果糖-1,6-二磷酸和ADP，丙酮酸激酶和乳酸脱氢酶进一步催化NADH氧化生成NAD⁺，在340nm下测定NADH下降速率，即可反映PFK活性。
适用样本：动植物组织、细胞、细胞上清、细菌、血清（浆）
| P1501170 | Component | 48T | 96T | Storage |
| P1501170A | Extraction Buffer | 60 mL | 60 mL×2 | 2-8℃ |
| P1501170B | Assay Buffer | 11 mL | 22 mL | 2-8℃ |
| P1501170C | Substrate Mix | 1EA | 1EA | 2-8℃. Store in the dark. |
| P1501170D | Enzyme 1 | 600 μL | 1.2 mL | -20℃. Store in the dark. |
| P1501170E | Enzyme 2 | 600 μL | 1.2 mL | -20℃. Store in the dark. |
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 孔酶标板 | UV板 |
| 试剂 | PBS (pH7.4) | 清洗样本用 |
| 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 |
| Working Substrate Mix | 临用前配制，加入Assay Buffer和去离子水充分溶解；48T加入10.2mL Assay Buffer 和0.678mL 去离子水，96T加入20.4mL Assay Buffer 和1.356mL 去离子水。 | 实验过程中避光放置； 未用完的Working Substrate Mix分装后-20℃避光保存2 周，避免反复冻融。 |
| Enzyme 1 | 即用型；使用前平衡到室温 | 实验过程中避光放置； -20℃避光保存 |
| Enzyme 2 | 使用前平衡到室温 | 实验过程中避光放置； -20℃避光保存 |
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。对于脂肪含量较高的动物组织，离心后移除上层脂肪，再取上清液。
2.1 动物组织：
称取约0.1g动物组织样本，加入1mL Extraction Buffer，冰浴匀浆，8000g，4℃离心10min，取上清液于新离心管中，置冰上待检测。
2.2 植物组织：
称取约0.1g植物组织样本，加入1mL Extraction Buffer捣碎，冰浴超声波破碎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 细胞或细菌：
收集5×10⁶个细胞或细菌，用冷的PBS清洗细胞或细菌，800g离心2min后弃上清，加入1mL Extraction Buffer，冰浴超声波破碎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。
2.4 血清（浆）、细胞上清：
直接测定。
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 Enzyme 1、10µL Enzyme 2和170µL Working Substrate Mix，混匀。
4.3 吸光度检测：立即记录340nm处20s时的吸光值A1和10min 20s后的吸光值A2。
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=A1-A2。
4.2 样本PFK活性计算
(1) 按样本质量计算
单位的定义：每g组织在反应体系中每分钟催化1nmol果糖-6-磷酸和1nmol ATP转化为1nmol果糖-1,6 二磷酸和1nmol ADP定义为一个酶活力单位。
PFK (U/g 鲜重)=[ΔA×V 反总 ÷(ε×d)×10⁹]÷(V 样 ÷V 样总 ×W)÷T=643×ΔA÷W
(2) 按细胞或细菌数量计算
单位的定义：每10⁴个细胞或细菌在反应体系中每分钟催化1nmol果糖-6-磷酸和1nmol ATP转化为1nmol 果糖-1,6-二磷酸和1nmol ADP定义为一个酶活力单位。
PFK (U/10⁴)=[ΔA×V 反总 ÷(ε×d)×10⁹]÷(V 样 ÷V 样总 ×N)÷T=643×ΔA÷N
(3) 按液体体积计算
单位的定义：每mL血清（浆）、细胞上清在反应体系中每分钟催化1nmol果糖-6-磷酸和1nmol ATP转化为1nmol 果糖-1,6-二磷酸和1nmol ADP定义为一个酶活力单位。
PFK (U/mL)=[ΔA×V 反总 ÷(ε×d)×10⁹]÷V 样 ÷T=643×ΔA
(4) 按蛋白浓度数量计算
单位的定义：每mg组织蛋白在反应体系中每分钟催化1nmol果糖-6-磷酸和1nmol ATP转化为1nmol 果糖-1,6-二磷酸和1nmol ADP定义为一个酶活力单位。
PFK (U/mg prot)=[ΔA×V 反总 ÷(ε×d)×10⁹]÷(Cpr×V 样 )÷T=643×ΔA÷Cpr
参数说明
V 反总 ：反应体系总体积，2.0×10⁻⁴ L；
ε：NADH摩尔消光系数，6.22×10³ mol/L/cm；
d：96孔板光径，0.5cm；
10⁹：1mol=1×10⁹ nmol；
V 样 ：加入样本体积，0.01 mL；
V 样总 ：加入Extraction Buffer体积，1 mL；
W：样品质量，g；
T：反应时间，10min；
Cpr：样本蛋白质浓度，mg/mL；
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：取0.1g玉米种子，按照测定步骤操作，用96孔UV板进行检测。测得ΔA=A1-A2=0.6772-0.6131 =0.0641，按样本质量计算得：
PFK(U/g 鲜重)=[ΔA×V 反总 ÷(ε×d)×10⁹]÷(V 样 ÷V 样总 ×W)÷T=643×ΔA÷W=411.5 U/g 鲜重。
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。
Phosphofructokinase (PFK, EC 2.7.1.11) is widely present in animals, plants, microorganisms, and cultured cells. It catalyzes the conversion of fructose-6-phosphate and ATP to fructose-1,6-bisphosphate and ADP, serving as one of the key regulatory enzymes in the glycolysis process.
Detection Principle: PFK catalyzes the conversion of fructose-6-phosphate and ATP to fructose-1,6-bisphosphate and ADP. Pyruvate kinase and lactate dehydrogenase further catalyze the oxidation of NADH to generate NAD⁺. The PFK activity is reflected by measuring the rate of decrease in NADH at 340 nm.
Applicable Samples: Animal/plant tissues, cells, cell supernatant, bacteria, serum (plasma).
| P1501170 | Component | 48T | 96T | Storage |
| P1501170A | Extraction Buffer | 60 mL | 60 mL×2 | 2-8℃ |
| P1501170B | Assay Buffer | 11 mL | 22 mL | 2-8℃ |
| P1501170C | Substrate Mix | 1EA | 1EA | 2-8℃. Store in the dark. |
| P1501170D | Enzyme 1 | 600 μL | 1.2 mL | -20℃. Store in the dark. |
| P1501170E | Enzyme 2 | 600 μL | 1.2 mL | -20℃. Store in the dark. |
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 Microplate | UV-transparent plate. |
| Reagents | PBS (pH 7.4) | For washing samples. |
| 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. |
| Working Substrate Mix | Prepare before use: For 48T: Add 10.2 mL Assay Buffer and 0.678 mL deionized water to the vial. For 96T: Add 20.4 mL Assay Buffer and 1.356 mL deionized water to the vial. Dissolve thoroughly. | Protect from light during the experiment. Unused Working Substrate Mix can be aliquoted and stored at -20°C protected from light for 2 weeks. Avoid repeated freeze-thaw cycles. |
| Enzyme 1 | Ready-to-use; equilibrate to room temperature before use. | Protect from light during the experiment; store at -20°C protected from light. |
| Enzyme 2 | Ready-to-use; equilibrate to room temperature before use. | Protect from light during the experiment; store at -20°C protected from light. |
2. Sample Preparation
Note: Fresh samples are recommended. If not used immediately, samples can be stored at -80°C for up to 1 month. For animal tissues with high fat content, remove the upper fat layer after centrifugation before collecting the supernatant.
2.1 Animal 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 into a new tube and keep on ice for detection.
2.2 Plant Tissues: Weigh approximately 0.1 g of tissue, add 1 mL of Extraction Buffer, and grind. 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 Cells or Bacteria: Collect 5×10⁶ cells or bacteria. Wash with cold PBS, centrifuge at 800 g for 2 min, 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.
2.4 Serum (Plasma), Cell Supernatant: 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 Enzyme 1, 10 µL of Enzyme 2, and 170 µL of Working Substrate Mix. Mix well.
3.3 Absorbance Measurement: Immediately record the absorbance at 340 nm at 20 seconds (A1) and then again after 10 minutes and 20 seconds (A2). Calculate ΔA = A1 - A2.
4. Result Calculation
The following provides both the derived formula and the simplified calculation formula, which are completely equivalent.
4.1 Data Processing
Calculate ΔA = A1 - A2.
4.2 Sample PFK Activity Calculation
(1) Based on sample mass:
Unit Definition: One unit of enzyme activity is defined as the amount that catalyzes the conversion of 1 nmol fructose-6-phosphate and 1 nmol ATP to 1 nmol fructose-1,6-bisphosphate and 1 nmol ADP per minute per gram of tissue in the reaction system.
Formula:
PFK (U/g fresh weight) = [ΔA × V total reaction ÷ (ε × d) × 10⁹] ÷ (V sample ÷ V total extract × W) ÷ T = 643 × ΔA ÷ W
(2) Based on cell or bacterial count:
Unit Definition: One unit of enzyme activity is defined as the amount that catalyzes the conversion of 1 nmol fructose-6-phosphate and 1 nmol ATP to 1 nmol fructose-1,6-bisphosphate and 1 nmol ADP per minute per 10⁴ cells or bacteria in the reaction system.
Formula:
PFK (U/10⁴) = [ΔA × V total reaction ÷ (ε × d) × 10⁹] ÷ (V sample ÷ V total extract × N) ÷ T = 643 × ΔA ÷ N
(3) Based on liquid volume:
Unit Definition: One unit of enzyme activity is defined as the amount that catalyzes the conversion of 1 nmol fructose-6-phosphate and 1 nmol ATP to 1 nmol fructose-1,6-bisphosphate and 1 nmol ADP per minute per mL of serum (plasma) or cell supernatant in the reaction system.
Formula:
PFK (U/mL) = [ΔA × V total reaction ÷ (ε × d) × 10⁹] ÷ V sample ÷ T = 643 × ΔA
(4) Based on protein concentration:
Unit Definition: One unit of enzyme activity is defined as the amount that catalyzes the conversion of 1 nmol fructose-6-phosphate and 1 nmol ATP to 1 nmol fructose-1,6-bisphosphate and 1 nmol ADP per minute per mg of protein in the reaction system.
Formula:
PFK (U/mg prot) = [ΔA × V total reaction ÷ (ε × d) × 10⁹] ÷ (Cpr × V sample ) ÷ T = 643 × ΔA ÷ Cpr
Parameter Description:
V total reaction : Total reaction volume, 2.0 × 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)
V sample : Volume of sample added, 0.01 mL
V total extract : Volume of Extraction Buffer added, 1 mL
W: Sample mass, g
T: Reaction time, 10 min
Cpr: Sample protein concentration, mg/mL
N: Cell or bacterial count, in units of 10⁴ (e.g., for 5×10⁶ cells, N=500)
5. Result Presentation
Example: 0.1 g of corn seeds was processed and assayed according to the procedure using a 96-well UV plate.
Measured: ΔA = A1 - A2 = 0.6772 - 0.6131 = 0.0641.
Calculated based on sample mass:
PFK (U/g fresh weight) = 643 × ΔA ÷ W = 411.5 U/g fresh weight.
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 > 0.5, the PFK activity in the sample is too high. Dilute the sample appropriately with Extraction Buffer or reduce the amount of sample used for extraction, and re-assay. If the sample ΔA is < 0.002, increase the sample amount.
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| 批号(Lot Number) | 证书类型 | 货号 |
|---|---|---|
 ZJ25F1028978 |
分析证书 | P1501170 |
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