BioReagent 级 ，适用于对基线干扰要求严格的色谱和分析工作流程。
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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。
NAD激酶（NADK,EC2.7.1.23）广泛存在于动物、植物、微生物和细胞中，是目前发现的生物体内唯一能够催化NAD+磷酸化生成NADP⁺的酶，可催化NAD(H)以ATP或无机多聚磷酸[poly(P)]作为磷酰基供体进行磷酸化反应，生成NADP(H)。因此，NADK在合成NADP(H)以及调节NAD(H)与NADP(H)的平衡上具有重要作用。
检测原理：在测定中，样本中存在的NADK催化NAD+磷酸化，生成NADP⁺；NADP⁺可被6-磷酸葡萄糖脱氢酶还原为NADPH，NADPH在340nm处有特征吸收峰，通过在340nm下测定NADPH增加速度（吸光值的变化）可反映出NADK活性。
适用样本：血清（浆）、动植物组织、细胞、细菌
| N1501164 | Component | 48T | 96T | Storage |
| N1501164A | Extraction Buffer | 60 mL | 60 mL×2 | 2-8℃ |
| N1501164B | Assay Buffer Ⅰ | 6 mL | 12 mL | 2-8℃ |
| N1501164C | Assay Buffer Ⅱ | 15 mL | 30 mL | 2-8℃ |
| N1501164D | Substrate Mix | 1EA | 1EA | -20℃. Store in the dark. |
| N1501164E | Enzyme Mix | 1EA | 1EA | -20℃. Store in the dark. |
注意：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。
自备仪器和试剂
酶标仪或紫外分光光度计（能测340nm处的吸光度）
96孔UV板或微量石英比色皿、可调节式移液枪及枪头
低温离心机、恒温箱、水浴锅
去离子水
匀浆器（如果是组织样本）
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 BufferⅠ | 即用型；使用前平衡到室温 | 4℃保存 |
| Assay BufferⅡ | 即用型；使用前平衡到室温 | 4℃保存 |
| Working ReagentⅠ | 使用前配制，在Substrate Mix中加入3mL (48T) / 6mL (96T) Assay BufferⅠ，充分混匀待用。 | 未使用的试剂分装后-20℃避光保存一周，避免反复冻融。 |
| Working ReagentⅡ | 使用前配制，在Enzyme Mix中加入12.6mL (48T) /25.2mL (96T) Assay BufferⅡ，充分混匀待用。 | 未使用的试剂分装后-20℃避光保存一周，避免反复冻融。 |
| Substrate Mix | 在测定过程中都必须在冰上放置。 | |
| Enzyme Mix | 在测定过程中都必须在冰上放置。 |
测定反应的温度对测定结果有影响，请控制在25℃（一般物种）或者37℃（哺乳动物）。
2、样本制备
2.1 血浆和血清样本：
可直接检测。
2.2 组织样本：
用冷的PBS冲洗组织去除组织表面的血液。称取约0.1g样本，加入1mL Extraction Buffer，冰浴匀浆，8,000g，4℃离心10min，取上清液，置冰上待测。
2.3 细胞和细菌样本：
收集500万细菌或细胞到离心管内，用冷PBS清洗，离心后弃上清，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8,000g，4℃离心10min，取上清液，置冰上待测。
2.4 植物样本：
用冷的PBS冲洗植物样本去除表面的杂质。称取约0.1g样本，加入1mL Extraction Buffer，冰浴匀浆，8,000g，4℃离心10min，取上清液，置冰上待测。
注意：如需测定蛋白浓度，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、实验步骤
3.1 酶标仪或紫外分光光度计预热30min以上，调节波长到340nm，紫外分光光度计用去离子水调零。
3.2 将Assay Buffer Ⅰ 37℃（哺乳动物）或25℃（其它物种）水浴15min以上。
3.3 设置测定管和对照管，按照如下方式加样（在EP管中操作）：
| 试剂 | 对照管 (μL) | 测定管 (μL) |
| 样本 | 20 | 20 |
| Working Reagent Ⅰ | 0 | 80 |
| Assay Buffer Ⅰ | 80 | 0 |
充分混匀，37℃（哺乳动物）或25℃（其它物种）水浴15min，立即煮沸2min（盖紧，防止水分散失）冰浴冷却，10,000g， 室温离心10min。
3.4 取各管中上清40μL加入96孔UV板或微量石英比色皿中，分别加入Working Reagent Ⅱ 160μL，迅速混匀。
3.5 加完试剂混匀，室温静置15min，在340nm下测定吸光值，ΔA=A 测定 -A 对照。
注意：每个样本均需要做对照管。实验之前建议选择2-3个预期差异大的样本做预实验。如果样本的ΔA小于0.01，可适当增大样本量，如果样本的ΔA大于2.0，样本可用Extraction Buffer进一步稀释，再进行实验，乘以最终的稀释倍数。
4、结果计算
我们为您提供的计算公式，包括推导过程计算公式和简洁计算公式，两者完全相等。建议以加粗的简洁计算公式为最终计算公式。
用96孔UV板测定的计算公式如下
4.1 血清（浆）NADK活性的计算 单位的定义：每mL血清（浆）每分钟生成1nmol的NADP定义为一个酶活力单位。
NADK (nmol/min/mL)＝[ΔA×V 反总 ÷(ε×d)×10⁹ ]÷V 样 ÷T=107.18×ΔA
4.2 组织、细菌或细胞中NADK活性的计算
(1) 按样本蛋白浓度计算 单位的定义：每mg组织蛋白每分钟生成1nmol NADP定义为一个酶活力单位。
NADK (nmol/min/mg prot)=[ΔA×V 反总 ÷(ε×d)× 10⁹ ]÷(V 样 ×Cpr)÷T=107.18×ΔA÷Cpr
(2) 按样本鲜重计算
单位的定义：每g组织每分钟生成1nmol NADP定义为一个酶活力单位。
NADK (nmol/min/g 鲜重)=[ΔA×V 反总 ÷(ε×d)× 10⁹ ]÷(W×V 样 ÷V 样总 )÷T=107.18×ΔA÷W
(3) 按细菌或细胞密度计算
单位的定义：每1万个细菌或细胞每分钟生成1nmol NADP定义为一个酶活力单位。
NADK (nmol/min/10⁴ )=[ΔA×V 反总 ÷(ε×d)×10⁹]÷(500×V 样 ÷V 样总 )÷T=0.214×ΔA
参数说明
V 反总 ：反应体系总体积，1×10⁻⁴ L；
ε：NADPH摩尔消光系数，6.22×10³ L/mol/cm；
d：96孔UV板光径，0.5cm； 10⁹：1mol=10⁹ nmol；
V 样 ：加入样本体积，0.02mL；
V 样总 ：加入Extraction Buffer体积，1mL；
T：反应时间，15min；
Cpr：样本蛋白质浓度，mg/mL；
W：样本质量，g；
500：细菌或细胞总数，500万个。
使用微量石英比色皿测定的计算公式 将上述计算公式中的光径d=0.5cm调整为d=1cm进行计算即可。
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、如需测定蛋白浓度，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。
NAD kinase (NADK, EC 2.7.1.23) is widely present in animals, plants, microorganisms, and cells. It is currently the only enzyme discovered in organisms that can catalyze the phosphorylation of NAD⁺ to generate NADP⁺. It catalyzes the phosphorylation of NAD(H) using ATP or inorganic polyphosphate [poly(P)] as the phosphoryl donor to produce NADP(H). Therefore, NADK plays a crucial role in synthesizing NADP(H) and regulating the balance between NAD(H) and NADP(H).
Detection Principle: In the assay, NADK present in the sample catalyzes the phosphorylation of NAD⁺ to generate NADP⁺. NADP⁺ can then be reduced to NADPH by glucose-6-phosphate dehydrogenase. NADPH has a characteristic absorption peak at 340 nm. The NADK activity is reflected by measuring the rate of increase in NADPH (the change in absorbance) at 340 nm.
Applicable Samples: Serum (plasma), animal/plant tissues, cells, bacteria.
| N1501164 | Component | 48T | 96T | Storage |
| N1501164A | Extraction Buffer | 60 mL | 60 mL×2 | 2-8℃ |
| N1501164B | Assay Buffer Ⅰ | 6 mL | 12 mL | 2-8℃ |
| N1501164C | Assay Buffer Ⅱ | 15 mL | 30 mL | 2-8℃ |
| N1501164D | Substrate Mix | 1EA | 1EA | -20℃. Store in the dark. |
| N1501164E | Enzyme Mix | 1EA | 1EA | -20℃. Store in the dark. |
Note: It is recommended to perform preliminary experiments using 2-3 samples expected to have significant differences before formal testing.
User-Provided Instruments and Reagents
Microplate reader or UV spectrophotometer (capable of measuring absorbance at 340 nm)
96-well UV plate or micro quartz cuvettes, Adjustable pipettes and tips
Low-temperature centrifuge, Incubator, Water bath
Deionized water
Homogenizer (for tissue samples)
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 I | Ready-to-use; equilibrate to room temperature before use. | Store at 4°C. |
| Assay Buffer II | Ready-to-use; equilibrate to room temperature before use. | Store at 4°C. |
| Working Reagent I | Prepare before use: Add 3 mL (48T) / 6 mL (96T) of Assay Buffer I to the Substrate Mix vial. Mix thoroughly. | Unused reagent can be aliquoted and stored at -20°C protected from light for one week. Avoid repeated freeze-thaw cycles. |
| Working Reagent II | Prepare before use: Add 12.6 mL (48T) / 25.2 mL (96T) of Assay Buffer II to the Enzyme Mix vial. Mix thoroughly. | Unused reagent can be aliquoted and stored at -20°C protected from light for one week. Avoid repeated freeze-thaw cycles. |
| Substrate Mix | Must be kept on ice during the assay procedure. | |
| Enzyme Mix | Must be kept on ice during the assay procedure. |
Note: The temperature of the assay reaction affects the results. Please control it at 25°C (for general species) or 37°C (for mammals).
2. Sample Preparation
2.1 Plasma and Serum Samples: Can be detected directly.
2.2 Tissue Samples: Rinse the tissue with cold PBS to remove blood from the surface. Weigh approximately 0.1 g of tissue, add 1 mL of Extraction Buffer, and homogenize on ice. Centrifuge at 8,000 g, 4°C for 10 minutes. Collect the supernatant and keep on ice for detection.
2.3 Cell and Bacterial Samples: Collect 5 million bacteria or cells 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 8,000 g, 4°C for 10 minutes. Collect the supernatant and keep on ice for detection.
2.4 Plant Samples: Rinse the plant sample with cold PBS to remove surface impurities. Weigh approximately 0.1 g of tissue, add 1 mL of Extraction Buffer, and homogenize on ice. Centrifuge at 8,000 g, 4°C for 10 minutes. Collect the supernatant and keep on ice for detection.
Note: For protein concentration determination, Aladdin BCA Protein Quantification Kit (B665595) or Ready-to-Use BCA Protein Quantification Kit (R1491648) are recommended.
3. Assay Steps
3.1 Instrument Preparation: Preheat the microplate reader or UV spectrophotometer for at least 30 minutes. Set the wavelength to 340 nm. For UV spectrophotometers, zero the instrument with deionized water.
3.2 Pre-incubate Assay Buffer I in a water bath at 37°C (for mammals) or 25°C (for other species) for at least 15 minutes.
3.3 Set up Test and Control tubes. Add reagents in EP tubes as follows:
| Reagent | Control Tube (µL) | Test Tube (µL) |
| Sample | 20 | 20 |
| Working Reagent I | 0 | 80 |
| Assay Buffer I | 80 | 0 |
Mix well and incubate in a water bath at 37°C (for mammals) or 25°C (for other species) for 15 minutes. Immediately boil for 2 minutes (cap tightly to prevent moisture loss). Cool in an ice bath. Centrifuge at 10,000 g, room temperature for 10 minutes.
3.4 Transfer 40 µL of the supernatant from each tube to a 96-well UV plate or micro quartz cuvette. Add 160 µL of Working Reagent II to each, and mix rapidly.
3.5 After adding reagents and mixing, let stand at room temperature for 15 minutes. Measure the absorbance at 340 nm. Calculate ΔA = A test - A control .
Note: A Control tube is required for each sample. It is recommended to perform preliminary experiments with 2-3 samples expected to have significant differences before formal testing. If the sample ΔA is less than 0.01, appropriately increase the sample amount. If the sample ΔA is greater than 2.0, dilute the sample further with Extraction Buffer and multiply the final result by the dilution factor.
4. Result Calculation We provide both derived and simplified calculation formulas, which are equivalent. The simplified formulas in bold are recommended as the final calculation formulas. Calculation formulas for using a 96-well UV plate:
4.1 NADK Activity in Serum (Plasma): Unit Definition: One unit of enzyme activity is defined as the generation of 1 nmol NADP per minute per mL of serum (plasma).
Formula:
NADK (nmol/min/mL) = [ΔA × V total reaction ÷ (ε × d) × 10⁹] ÷ V sample ÷ T = 107.18 × ΔA
4.2 NADK Activity in Tissues, Bacteria, or Cells:
(1) Based on sample protein concentration:
Unit Definition: One unit of enzyme activity is defined as the generation of 1 nmol NADP per minute per mg of protein.
Formula:
NADK (nmol/min/mg prot) = [ΔA × V total reaction ÷ (ε × d) × 10⁹] ÷ (V sample × Cpr) ÷ T = 107.18 × ΔA ÷ Cpr
(2) Based on sample fresh weight:
Unit Definition: One unit of enzyme activity is defined as the generation of 1 nmol NADP per minute per gram of fresh tissue.
Formula:
NADK (nmol/min/g fresh weight) = [ΔA × V total reaction ÷ (ε × d) × 10⁹] ÷ (W × V sample ÷ V total extract ) ÷ T = 107.18 × ΔA ÷ W
(3) Based on bacterial or cell density:
Unit Definition: One unit of enzyme activity is defined as the generation of 1 nmol NADP per minute per 10⁴ bacteria or cells.
Formula:
NADK (nmol/min/10⁴) = [ΔA × V total reaction ÷ (ε × d) × 10⁹] ÷ (500 × V sample ÷ V total extract ) ÷ T = 0.214 × ΔA
Parameter Description:
V total reaction : Total reaction volume, 1 × 10⁻⁴ L
ε: Molar extinction coefficient of NADPH, 6.22 × 10³ L/mol/cm
d: Light path of the 96-well UV plate, 0.5 cm
10⁹: Conversion factor (1 mol = 10⁹ nmol)
V sample : Volume of sample added, 0.02 mL
V total extract : Volume of Extraction Buffer added, 1 mL
T: Reaction time, 15 min Cpr: Sample protein concentration, mg/mL
W: Sample mass, g
500: Total number of bacteria or cells, 5 million
Calculation formulas for using micro quartz cuvettes:
Adjust the light path in the formulas above from d=0.5 cm to d=1 cm.
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 protein concentration determination, 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.
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| 批号(Lot Number) | 证书类型 | 货号 |
|---|---|---|
 ZJ25F1028977 |
分析证书 | N1501164 |
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