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。
烟酰胺腺嘌呤二核苷酸磷酸（Nicotinamideadeninedinucleotide phosphate，NADP）是很多氧化还原反应的辅酶，包括NADP+（氧化型）和NADPH（还原型）两种形式。NADP+也参与到生物合成反应中，比如脂质和核酸的合成。在动物细胞中，戊糖磷酸途径（pentose phosphate pathway，PPP）的氧化阶段是NADPH最主要的来源。NADP+/NADPH的测定主要应用于有关细胞或组织能量转化和氧化还原状态的研究中。
检测原理：葡萄糖脱氢酶循环反应（不识别NAD+/NADH），在这一反应过程中生成的NADPH 将WST-8还原生成橙黄色的formazan（甲臜），在450nm左右检测有最大吸收峰，反应体系中生成的formazan与样品中总的NADP+或NADPH的总量呈正比关系。
检测范围：0.5-20 µM
灵敏度：0.5µM
适用样本：动植物组织、细胞、细菌、血清（浆）
| Component | 48T | 96T | Storage |
| Assay Buffer | 7.5 mL | 15 mL | 2-8℃ |
| Glucose (1M) | 1 mL | 2 mL | 2-8℃ |
| WST-8 | 350 μL | 700 μL | -20℃. Store in the dark. |
| Enhancer | 70 µL | 140 µL | -20℃. Store in the dark. |
| NADP Cycling Enzyme Mix | 70 µL | 140 µL | -20℃. Store in the dark. |
| NADPH Standard | 1EA | 1EA | -20℃. Store in the dark. |
| NADP Extraction Buffer | 7 mL | 14 mL | 2-8℃ |
| NADPH Extraction Buffer | 7 mL | 14 mL | 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450nm处的吸光度 |
| 耗材 | 96孔酶标板 | 普通透明板 |
| 试剂 | PBS（pH7.4）、去离子水 | 清洗/稀释样本用 |
| 其他 | 匀浆器（组织样本）、恒温箱、制冰机、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 |
| Assay Buffer | 即用型；使用前平衡到室温 | 4℃保存 |
| Glucose (1M) | 即用型；使用前平衡到室温 | 4℃保存 |
| WST-8 | 即用型 | 实验过程中，冰上避光放置；分装避光保存于-20℃ |
| Enhancer | 即用型 | 实验过程中，冰上避光放置；分装避光保存于-20℃ |
| NADP Cycling Enzyme Mix | 即用型 | 实验过程中，冰上避光放置；分装避光保存于-20℃ |
| NADPH Standard | 临用前配制；加1mL的去离子水充分溶解，标准品的浓度 是10mM | 实验过程中，冰上避光放置；分装避光保存于-20℃ |
| NADP Extraction Buffer | 即用型；使用前平衡到室温 | 4℃保存 |
| NADPH Extraction Buffer | 即用型；使用前平衡到室温 | 4℃保存 |
2、标准品配制
取2µL 10mM的NADPH加998µL Assay Buffer稀释为1mL 20µM NADPH预混液。按下表所示，进 行下一步稀释:
| 标准品工作液 | 20µM NADPH预混液 (µL) | Assay Buffer (µL) | NADPH (µM) |
| 1 | 100 | 0 | 20 |
| 2 | 50 | 50 | 10 |
| 3 | 40 | 60 | 8 |
| 4 | 20 | 80 | 4 |
| 5 | 10 | 90 | 2 |
| 6 | 5 | 95 | 1 |
| 7 | 2.5 | 97.5 | 0.5 |
3、样品制备
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。当准备好进行该项实验时，将样本从-80℃拿出后，冰上解冻。但是请注意，这可能会影响样本的稳定性，实验结果可能会低于预期。为避免干扰实验，样本中应避免有如下物质存在：EDTA (>0.5mM)，ascorbic acid，SDS (>0.2%)，sodium azide，NP-40 (>1%)及Tween-20 (>1%)。
3.1 组织样本
冰上预冷的PBS洗涤组织后，称取约20mg的组织样品，用剪刀剪碎，置于匀浆器中。
提取NADP：加入100µL NADP Extraction Buffer匀浆样本。煮沸5min（盖紧，以防止水分散失）后加入20µL Assay Buffer和100µL NADPH Extraction Buffer中和提取物。4℃，14000rpm离心 5min。将上清转移到新离心管，冰上待测。
提取NADPH：加入100µL NADPH Extraction Buffer匀浆样本。煮沸5min（盖紧，以防止水分散失）后加入20µL Assay Buffer和100µL NADP Extraction Buffer中和提取物。4℃，14000rpm离心5min。将上清转移到新离心管，冰上待测。
3.2 细胞或细菌样本
收集1×10⁶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。
提取NADP：加入100µL NADP Extraction Buffer，超声波破碎5min（功率20%或200W，超声3s，间隔7s，重复30次），煮沸5min（盖紧，以防止水分散失）后加入20µL Assay Buffer和100µL NADPH Extraction Buffer中和提取物。4℃，14000rpm离心5min，将上清转移到新离心管，冰上待测。
提取NADPH：加入100µL NADPH Extraction Buffer，超声波破碎5min（功率20%或200W，超声3s， 间隔7s，重复30次），煮沸5min（盖紧，以防止水分散失）后加入20µL Assay Buffer和100µL NADP Extraction Buffer 中和提取物。4℃，14000rpm离心5min，将上清转移到新离心管，冰上待测。
3.3 血清（浆）样本
提取NADP：吸取约20µL的血清（浆），加入100µL NADP Extraction Buffer，煮沸5min（盖紧，以防止水分散失）后加入20µL Assay Buffer和100µL NADPH Extraction Buffer中和提取物。4℃，14000rpm离心5min，将上清转移到新离心管，冰上待测。
提取NADPH：吸取约20µL的血清（浆），加入100µL NADPH Extraction Buffer，煮沸5min（盖紧，以防止水分散失）后加入20µL Assay Buffer和100µL NADP Extraction Buffer中和提取物。4℃，14000rpm离心5min，将上清转移到新离心管，冰上待测。
4、实验步骤
4.1 酶标仪准备：预热30min以上，调节波长到450nm。
4.2 工作液的配制：每孔配制85µL工作液，现配现用。吸取68µL Assay Buffer，1µL NADP Cycling Enzyme Mix，5µL WST-8，1µL Enhancer混合后，室温孵育5min，再加入10µL Glucose。
4.3 检测体系配制：在96孔板中按照如下方式操作。该分析基于酶催化的动力学反应，工作液的加入需要迅速，各组分需要彻底混合。建议使用多通道移液器。标准孔和空白孔一般只做1次。
| 试剂 | 空白孔（μL） | 标准孔（μL） | 测定孔（μL） |
| Assay Buffer | 40 | 0 | 0 |
| 标准品工作液 | 0 | 40 | 0 |
| 样本 | 0 | 0 | 40 |
| 工作液 | 80 | 80 | 80 |
4.4 吸光度检测：充分混匀，测定450nm处的吸光值A1；室温孵育30min，再测定450nm处的吸光值A2。
5、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。
5.1 数据处理
计算ΔA=A2-A1，ΔΔA标准=ΔA标准-ΔA空白，ΔΔA测定=ΔA测定-ΔA空白。
5.2 标准曲线绘制
以标准品浓度为y轴，ΔΔA标准为x轴，绘制标准曲线。将ΔΔA测定代入方程得到y值（µM）。
5.3 样本NADP或NADPH含量计算
(1) 按样本质量计算
NADP或NADPH (nmol/g)=(y×V样)÷(W×V样÷V样总)×n=0.22×y÷W×n
(2) 按细胞或细菌数量计算
NADP或NADPH (nmol/10⁴)=(y×V样)÷(N×V样÷V样总)×n=0.22×y÷N×n
(3) 按液体体积计算
NADP或NADPH (nmol/mL)=y×(V样总+V提取)÷V提取×n=12×y×n
(4) 按蛋白浓度数量计算
NADP或NADPH (nmol/mg prot)=(y×V样)÷(V样×Cpr)×n=y÷Cpr×n
参数说明
nmol：1 μM=1 nmol/mL；
V样：加入样本体积，0.04mL；
V样总：组织样本总体积，0.22mL；
V提取：血清（浆） 的体积，0.02mL；
Cpr：样本蛋白质浓度，mg/mL；
W：样本质量，g；
N：细胞或细菌数量，以10⁴为单位 （例如细胞数量为1×10⁶，N=100）；
n：样本稀释倍数。
6、结果展示
典型标准曲线：y=26.294x+0.0516，R²=0.9998
注意：在这些浓度下，NADP和NADPH的标准曲线是相同的。此说明书中，我们仅提供NADPH标准曲线。
常见问题及解答
Q：检测得到的样本ΔA测定过高或者过低怎么办？
A：如果样本的ΔΔA测定值高于20µM标准品的ΔΔA标准值，将样本用去离子水进行稀释后，再进行实验。如果样本的ΔΔA测定值低于0.5µM标准品的ΔΔA标准值，增加样本量。
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、建议实验者自行建立标准曲线以提高结果准确度。如未自行建立标准曲线，可参考结果展示部分的典型标准曲线公式进行结果计算。
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。
Nicotinamide adenine dinucleotide phosphate (NADP) is a coenzyme for many oxidoreductases and exists in two forms: NADP⁺ (oxidized) and NADPH (reduced). NADP⁺ is also involved in biosynthetic reactions, such as the synthesis of lipids and nucleic acids. In animal cells, the oxidative phase of the pentose phosphate pathway (PPP) is the primary source of NADPH. The measurement of NADP⁺/NADPH is mainly applied in studies related to cellular or tissue energy conversion and redox status.
Assay Principle: This assay utilizes a glucose dehydrogenase cycling reaction (which does not recognize NAD⁺/NADH). The NADPH generated in this process reduces WST-8 to produce an orange-yellow formazan dye, which has a maximum absorption peak around 450 nm. The amount of formazan generated in the reaction system is proportional to the total amount of NADP⁺ or NADPH in the sample.
Detection Range: 0.5-20 µM
Sensitivity: 0.5 µM
Applicable Samples: Animal/plant tissues, cells, bacteria, serum/plasma
| Component | 48T | 96T | Storage |
| Assay Buffer | 7.5 mL | 15 mL | 2-8℃ |
| Glucose (1M) | 1 mL | 2 mL | 2-8℃ |
| WST-8 | 350 μL | 700 μL | -20℃. Store in the dark. |
| Enhancer | 70 µL | 140 µL | -20℃. Store in the dark. |
| NADP Cycling Enzyme Mix | 70 µL | 140 µL | -20℃. Store in the dark. |
| NADPH Standard | 1EA | 1EA | -20℃. Store in the dark. |
| NADP Extraction Buffer | 7 mL | 14 mL | 2-8℃ |
| NADPH Extraction Buffer | 7 mL | 14 mL | 2-8℃ |
Please check the volume of all components before the experiment.
An extra 10% volume is provided for standard curve preparation or pre-experiments.
User-Provided Instruments and Reagents
| Type | Name | Notes |
| Instrument | Microplate Reader | Capable of measuring absorbance at 450 nm |
| Consumables | 96-well microplate | Standard transparent plate |
| Reagents | PBS (pH 7.4), Deionized Water | For washing/diluting samples |
| Others | Homogenizer (for tissue samples), Incubator, Ice Maker, Refrigerated Centrifuge, Adjustable Pipettes and Tips | Using a multichannel pipette is recommended for high-throughput assays |
Experimental Procedure
1. Reagent Preparation
| Reagent Name | Preparation | Notes |
| Assay Buffer | Ready-to-use; equilibrate to RT before use | Store at 4°C |
| Glucose (1M) | Ready-to-use; equilibrate to RT before use | Store at 4°C |
| WST-8 | Ready-to-use | Keep on ice protected from light during experiment; store aliquoted at -20°C protected from light |
| Enhancer | Ready-to-use | Keep on ice protected from light during experiment; store aliquoted at -20°C protected from light |
| NADP Cycling Enzyme Mix | Ready-to-use | Keep on ice protected from light during experiment; store aliquoted at -20°C protected from light |
| NADPH Standard | Reconstitute with 1 mL deionized water before use to 10 mM concentration | Keep on ice protected from light during experiment; store aliquoted at -20°C protected from light |
| NADP Extraction Buffer | Ready-to-use; equilibrate to RT before use | Store at 4°C |
| NADPH Extraction Buffer | Ready-to-use; equilibrate to RT before use | Store at 4°C |
2. Standard Preparation
Pipette 2 µL of the 10 mM NADPH standard into 998 µL Assay Buffer to prepare 1 mL of 20 µM NADPH premix. Then dilute serially as shown in the table below:
| Standard Working Solution | Volume of 20 µM NADPH Premix (µL) | Volume of Assay Buffer (µL) | Final NADPH Concentration (µM) |
| 1 | 100 | 0 | 20 |
| 2 | 50 | 50 | 10 |
| 3 | 40 | 60 | 8 |
| 4 | 20 | 80 | 4 |
| 5 | 10 | 90 | 2 |
| 6 | 5 | 95 | 1 |
| 7 | 2.5 | 97.5 | 0.5 |
3. Sample Preparation
Note: Fresh samples are recommended. If not used immediately, samples can be stored at -80°C for up to 1 month. Thaw frozen samples on ice before use, noting that this may affect stability and result in lower than expected values. Avoid the presence of interfering substances such as: EDTA (>0.5 mM), ascorbic acid, SDS (>0.2%), sodium azide, NP-40 (>1%), and Tween-20 (>1%).
3.1 Tissue Samples
Rinse tissue with ice-cold PBS. Weigh about 20 mg of tissue, mince with scissors, and place in a homogenizer.
For NADP extraction: Add 100 µL NADP Extraction Buffer and homogenize. Boil for 5 min (tightly capped to prevent evaporation). Then add 20 µL Assay Buffer and 100 µL NADPH Extraction Buffer to neutralize the extract. Centrifuge at 4°C, 14,000 rpm for 5 min. Transfer the supernatant to a new tube and keep on ice for assay.
For NADPH extraction: Add 100 µL NADPH Extraction Buffer and homogenize. Boil for 5 min (tightly capped to prevent evaporation). Then add 20 µL Assay Buffer and 100 µL NADP Extraction Buffer to neutralize the extract. Centrifuge at 4°C, 14,000 rpm for 5 min. Transfer the supernatant to a new tube and keep on ice for assay.
3.2 Cell or Bacterial Samples
Collect 1×10⁶ cells/bacteria. Wash with cold PBS, centrifuge at 800 g for 2 min, and discard the supernatant.
For NADP extraction: Add 100 µL NADP Extraction Buffer. Sonicate for 5 min (20% power or 200 W, pulse 3s on, 7s off, repeat 30 times). Boil for 5 min (tightly capped). Then add 20 µL Assay Buffer and 100 µL NADPH Extraction Buffer to neutralize. Centrifuge at 4°C, 14,000 rpm for 5 min. Transfer the supernatant to a new tube and keep on ice for assay.
For NADPH extraction: Add 100 µL NADPH Extraction Buffer. Sonicate for 5 min (20% power or 200 W, pulse 3s on, 7s off, repeat 30 times). Boil for 5 min (tightly capped). Then add 20 µL Assay Buffer and 100 µL NADP Extraction Buffer to neutralize. Centrifuge at 4°C, 14,000 rpm for 5 min. Transfer the supernatant to a new tube and keep on ice for assay.
3.3 Serum/Plasma Samples
For NADP extraction: Pipette about 20 µL of serum/plasma. Add 100 µL NADP Extraction Buffer. Boil for 5 min (tightly capped). Then add 20 µL Assay Buffer and 100 µL NADPH Extraction Buffer to neutralize. Centrifuge at 4°C, 14,000 rpm for 5 min. Transfer the supernatant to a new tube and keep on ice for assay.
For NADPH extraction: Pipette about 20 µL of serum/plasma. Add 100 µL NADPH Extraction Buffer. Boil for 5 min (tightly capped). Then add 20 µL Assay Buffer and 100 µL NADP Extraction Buffer to neutralize. Centrifuge at 4°C, 14,000 rpm for 5 min. Transfer the supernatant to a new tube and keep on ice for assay.
4. Assay Procedure
4.1 Microplate Reader Preparation: Preheat for at least 30 minutes. Set the wavelength to 450 nm.
4.2 Working Solution Preparation: Prepare 85 µL working solution per well fresh. Mix 68 µL Assay Buffer, 1 µL NADP Cycling Enzyme Mix, 5 µL WST-8, and 1 µL Enhancer. Incubate at room temperature for 5 min. Then add 10 µL Glucose.
4.3 Assay System Setup: Pipette reagents into a 96-well plate as follows. This assay is based on an enzyme-catalyzed kinetic reaction; add working solution quickly and mix all components thoroughly. A multichannel pipette is recommended. Standard and Blank wells are typically set up in singlicate.
| Reagent | Blank Well (μL) | Standard Well (μL) | Test Well (μL) |
| Assay Buffer | 40 | 0 | 0 |
| Standard Working Solution | 0 | 40 | 0 |
| Sample | 0 | 0 | 40 |
| Working Solution | 80 | 80 | 80 |
4.4 Absorbance Measurement: Mix thoroughly. Measure the absorbance at 450 nm (A₁). Incubate at room temperature for 30 min. Measure the absorbance at 450 nm again (A₂).
5. Calculation of Results
The following derived formula and simplified formula are completely equivalent.
5.1 Data Processing
Calculate ΔA = A₂ - A₁.
Calculate ΔΔAstandard = ΔAstandard - ΔAblank
Calculate ΔΔAtest = ΔAtest - ΔAblank
5.2 Standard Curve Plotting
Plot the standard curve with the standard concentration (µM) on the Y-axis and ΔΔAstandard on the X-axis. Substitute ΔΔAtest into the equation to obtain the Y value (µM).
5.3 Sample NADP or NADPH Content Calculation
(1) Based on sample weight:
NADP or NADPH (nmol/g weight) = (Y × Vsample) ÷ (W × Vsample ÷ Vtotal) × n = 0.22 × Y ÷ W × n
(2) Based on cell/bacteria count:
NADP or NADPH (nmol/10⁴ cells) = (Y × Vsample) ÷ (N × Vsample ÷ Vtotal) × n = 0.22 × Y ÷ N × n
(3) Based on liquid volume:
NADP or NADPH (nmol/mL) = Y × (Vtotal + Vextract) ÷ Vextract × n = 12 × Y × n
(4) Based on protein concentration:
NADP or NADPH (nmol/mg prot) = (Y × Vsample) ÷ (Vsample × Cpr) × n = Y ÷ Cpr × n
Parameter Definitions:
nmol: 1 µM = 1 nmol/mL;
Vsample: Volume of sample added to well, 0.04 mL;
Vtotal: Total volume of tissue sample extract, 0.22 mL;
Vextract: Volume of serum/plasma used for extraction, 0.02 mL;
Cpr: Sample protein concentration, mg/mL;
W: Sample weight, g;
N: Cell/Bacteria number, in units of 10⁴ (e.g., if cell number is 1×10⁶, N=100);
n: Sample dilution factor.
6. Typical Results
Typical Standard Curve: Y=26.294X+0.0516, R²=0.9998
Note: The standard curves for NADP and NADPH are identical at these concentrations. This manual provides only the NADPH standard curve.
FAQ
Q: What should I do if the sample ΔAtest is too high or too low?
A: If the sample ΔΔAtest is higher than the ΔΔAstandard of the 20 µM standard, dilute the sample with deionized water before repeating the assay. If the sample ΔΔAtest is lower than the ΔΔAstandard of the 0.5 µM standard, increase the sample amount used for extraction.
Notes
1. It is recommended to perform a pilot experiment using 2-3 samples expected to have significant differences before formal testing.
2. For tissue and cell samples, results can be normalized by measuring protein concentration. For protein concentration determination, Aladdin BCA Protein Quantification Kit (B665595) or Ready-to-Use BCA Protein Quantification Kit (R1491648) are recommended.
3. This kit is compatible with spectrophotometer detection. Adjust the preparation volume of detection reagents proportionally according to the spectrophotometer's requirements.
4. It is recommended to establish a standard curve for each experiment to improve accuracy. If a standard curve is not run, refer to the typical standard curve formula in the 'Results' section for calculation.
5. Biochemical reagents are generally irritating and/or toxic. For your safety and health, please wear appropriate personal protective equipment (lab coat, mask, gloves, head cover, etc.) throughout the experiment and perform steps in a fume hood or biosafety cabinet when applicable.
6. This product is for research use only. Not for use in diagnostic procedures.
危险品化学品经营许可证（带存储）