NADPH、 NADP⁺ 作为细胞核心氧化还原辅酶，参与核酸合成、脂质代谢、抗氧化防御、ROS 清除及细胞凋亡调控，二者比值直接反映细胞氧化还原平衡与代谢重编程状态。肿瘤发生、机体衰老、神经损伤、炎症应激、动植物抗逆等众多生理病理过程，均伴随着辅酶稳态紊乱，精准定量二者含量与比值，是代谢机制研究、疾病靶点探索、药效筛选不可或缺的检测手段。
本试剂盒基于特异性酶循环显色原理，可高效分离区分 NADPH 与 NADP⁺ 有效规避 NADH/ NAD⁺ 内源物质干扰，检测灵敏度高、重复性稳定。检测灵敏度高，线性范围宽，样品用量少。在样品体积为20 µL时，采用吸光度法检测在0.03-4μM浓度范围内有良好的线性关系。
N1375254 | 组件 | 50 T | 100 T | 储存条件 | N1375254A | 葡萄糖-6-磷酸脱氢酶 | 100µL | 200uL | -20℃.避光. | N1375254B | 葡萄糖-6-磷酸 | 4.9mL | 9.8mL | -20℃.避光. | N1375254C | NADPH | 5mg | 10mg | -20℃.避光. | N1375254D | NADPH 稀释液 | 10mL | 20mL | -20℃. | N1375254E | 显色液 | 200µL | 400µL | -20℃.避光. | N1375254F | NADP⁺ 提取液 | 10mL | 20mL | -20℃. | N1375254G | NADPH 提取液 | 10mL | 20mL | -20℃. |
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使用方法
1. NADP⁺/NADPH提取
（1）细胞或细菌样品的提取：对于贴壁细胞，弃掉培养基，用胰酶消化或适当拍打使细胞脱离培养器皿并转移至离心管中，用PBS 重悬洗涤一次，离心 (400-500×g, 5分钟)收集细胞到离心管内，弃上清并吸净残留液体。对于培养的悬浮细胞，离心 (400-500×g, 5分钟)收集细胞到离心管内，PBS 重悬洗涤一次，离心 (400-500×g, 5分钟)收集细胞，弃上清并吸净残留液体。
NADP⁺提取：每100万个细胞加入100-200μL NADP提取液，适当吹打后4℃或冰浴等低温条件下进行超声破碎细胞3min(200W，超声2s，停1s)。超声结束后，50℃加热5min，加热结束后，放置冰上冷却，待冷却后，再加入等体积的 NADPH提取液混匀中和，4ºC, 12,000×g离心3分钟，取上清用于后续检测。
NADPH提取：每100万个细胞加入100-200μL NADPH提取液，适当吹打后4℃或冰浴等低温条件下进行超声破碎细胞3min(200W，超声2s，停1s)。超声结束后，50℃加热5min，加热结束后，放置冰上冷却，待冷却后，再加入等体积的 NADP⁺提取液混匀中和，4ºC, 12,000×g离心3分钟，取上清用于后续检测。
（2）组织样品的提取
NADP⁺提取：每10 mg组织加入100 -200μL NADP⁺提取液，适当吹打后4℃或冰浴等低温条件下进行超声破碎细胞。超声结束后，50℃加热5min，加热结束后，放置冰上冷却，待冷却后，再加入等体积的 NADPH提取液混匀中和，4ºC, 12,000×g离心3分钟，取上清用于后续检测。
NADPH提取：每10 mg组织加入100 -200μL NADPH提取液，适当吹打后4℃或冰浴等低温条件下进行超声破碎细胞。超声结束后，50℃加热5min，加热结束后，放置冰上冷却，待冷却后，再加入等体积的 NADP⁺提取液混匀中和，4ºC, 12,000×g离心3分钟，取上清用于后续检测。
注意：以上所有操作均需在4ºC或冰上操作。制备好的细胞或组织样品如果不能立即检测，可以-20ºC或-80ºC冻存。
2. 显色工作液的配制
首先将稀释液和提取液平衡至室温，然后按照每个检测反应104 μL的体积配制显色工作液。根据待检测样品(包括标准品)的数量，配制适量的显色工作液。显色工作液的各个组份的具体配制方法参考下表。由于显色液室温容易见光分解，建议尽量现配现用，如果不能及时检测，请置于4℃或者冰浴避光保存且不要过夜，以免影响检测效果。
样本数 | 1 | 10 | 20 | 50 | G6P | 98 μL | 980 μL | 1960 μL | 4900 μL | G6PD | 2 μL | 20 μL | 40 μL | 100 μL | 显色液 | 4 μL | 40 μL | 80 μL | 200 μL |
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3. 样本测定
标准曲线绘制：
用6mL超纯水将本试剂盒提供的5mg的NADPH粉末全部溶解，配置成浓度为1mM的NADPH标准品溶液，为避免反复冻融，请分装后放置-80℃避光保存。用NADPH稀释液将1mM的NADPH标品溶液分别稀释至4μM，2μM，1μM，0.5μM，0.25μM，0.125μM，0.0625μM，0.03125uM和0μM。其中0μM为空白孔，仅含NADPH稀释液。取20 μL不同浓度的标品溶液至96孔板孔中，然后每孔添加104μL显色工作液，混匀后，于37℃反应10min后读取A450。
样品测定
取20 μL样品或稀释后的样品至96孔板样品孔中，然后每孔加入添加104μL显色工作液，混匀后，于37℃反应10min后读取A450。
4. 结果计算
建立标准曲线时，需要减去空白孔对照。标准曲线建立完毕后，可根据标准曲线计算出样品中的NADPH浓度（C），计算公示如下：
C(μM)=A×2n
注：A为根据标准曲线计算出的NADPH浓度(μM)；2n为样品稀释倍数再乘以2。因为样品处理完后加入等体积的NADP或NADPH中和液，相当多稀释了一倍。
注意事项
1. NADPH不稳定，建议标品现用现配，且注意避光。
2. 显色工作液同样建议尽量现配现用。
3. NADP或NADPH提取液具有一定腐蚀性，请一定要规范穿戴实验服和佩戴橡胶手套，以免接触皮肤。
As core cellular redox coenzymes, NADPH and NADP+ participate in nucleic acid synthesis, lipid metabolism, antioxidant defense, ROS scavenging, and the regulation of cell apoptosis. The ratio of NADPH to NADP+ directly reflects the cellular redox homeostasis and metabolic reprogramming status. A wide range of physiological and pathological processes, including tumorigenesis, organismal aging, nerve injury, inflammatory stress, and plant/animal stress tolerance, are accompanied by the disturbance of coenzyme homeostasis. Accurate quantification of the contents and ratio of NADPH and NADP+ serves as an indispensable detection approach for metabolic mechanism research, disease target exploration, and drug efficacy screening.
Based on the specific enzymatic cycling colorimetric principle, this assay kit can efficiently separate and differentiate NADPH from NADP+, effectively avoiding the interference of endogenous NADH/NAD+. It features high detection sensitivity, excellent repeatability, a broad linear range, and low sample consumption. With a sample volume of 20 µL, the absorbance-based detection exhibits an ideal linear relationship within the concentration range of 0.03–4 μM.
N1375254 | Components | 50 T | 100 T | Storage | N1375254A | G6PD | 100µL | 200uL | -20℃. dark . | N1375254B | G6P | 4.9mL | 9.8mL | -20℃. dark . | N1375254C | NADPH | 5mg | 10mg | -20℃. dark . | N1375254D | NADP Dilution Buffer | 10mL | 20mL | -20℃. | N1375254E | Chromogenic solution | 200µL | 400µL | -20℃. dark . | N1375254F | NADP+ Extraction Buffer | 10mL | 20mL | -20℃. | N1375254G | NADPH Extraction Buffer | 10mL | 20mL | -20℃. |
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Usage Method
1. Extraction of NADP+/NADPH
(1) Extraction from Cell or Bacterial Samples
For adherent cells: Discard the culture medium. Detach cells from the culture vessel by trypsin digestion or gentle tapping, then transfer the cell suspension into a centrifuge tube. Resuspend and wash cells once with PBS, then centrifuge at 400–500×g for 5 min to pellet the cells. Discard the supernatant and completely remove residual liquid.
For suspension cells in culture: Centrifuge the cell culture at 400–500×g for 5 min to collect cells into a centrifuge tube. Resuspend and wash once with PBS, recentrifuge at 400–500×g for 5 min, discard the supernatant and aspirate off all residual liquid.
NADP+ Extraction: Add 100–200 μL of NADP+ extraction solution per 1×10⁶ cells. Pipette gently to mix, then perform ultrasonic cell lysis for 3 min under low-temperature conditions (4 °C or ice bath) with the following parameters: 200 W, 2 s ultrasonication followed by 1 s interval. After ultrasonication, heat the sample at 50 °C for 5 min, then place it on ice to cool down. After cooling, add an equal volume of NADPH extraction solution, mix thoroughly for neutralization, centrifuge at 12,000×g for 3 min at 4 °C, and collect the supernatant for subsequent detection.
NADPH Extraction: Add 100–200 μL of NADPH extraction solution per 1×10⁶ cells. Pipette gently to mix, then perform ultrasonic cell lysis for 3 min under low-temperature conditions (4 °C or ice bath) with parameters: 200 W, 2 s ultrasonication / 1 s interval. After ultrasonication, heat at 50 °C for 5 min and cool on ice. Subsequently add an equal volume of NADP+ extraction solution and mix for neutralization. Centrifuge at 12,000×g for 3 min at 4 °C, and retain the supernatant for subsequent assays.
(2) Extraction from Tissue Samples
NADP+ Extraction: Add 100–200 μL of NADP extraction solution per 10 mg tissue. Pipette gently, then lyse the tissue by ultrasonication at low temperature (4 °C or ice bath). After ultrasonication, heat at 50 °C for 5 min and cool on ice. Add an equal volume of NADPH extraction solution, mix well to neutralize, centrifuge at 12,000×g for 3 min at 4 °C, and collect the supernatant for subsequent detection.
NADPH Extraction: Add 100–200 μL of NADPH extraction solution per 10 mg tissue. Pipette gently, then perform ultrasonic lysis at low temperature (4 °C or ice bath). After ultrasonication, heat at 50 °C for 5 min and cool on ice. Add an equal volume of NADP+ extraction solution, mix thoroughly for neutralization, centrifuge at 12,000×g for 3 min at 4 °C, and collect the supernatant for subsequent detection.
Note: All operations above must be performed at 4 °C or on ice. If the prepared cell or tissue samples cannot be assayed immediately, they can be stored frozen at −20 °C or −80 °C.
2. Preparation of Chromogenic Working Solution
First equilibrate the detection buffer to room temperature. Prepare the chromogenic working solution at a volume of 104 μL per reaction. Prepare an appropriate amount according to the number of test samples (including standards). Refer to the table below for the detailed formulation of each component in the chromogenic working solution.
Number of sample | 1 | 10 | 20 | 50 | G6P | 98 μL | 980 μL | 1960 μL | 4900 μL | G6PD | 2 μL | 20 μL | 40 μL | 100 μL | Chromogenic Solution | 4 μL | 40 μL | 80 μL | 200 μL |
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The chromogenic solution is prone to photodegradation at room temperature; it is recommended to prepare it immediately before use. If assay cannot be performed promptly, store the solution at 4 °C or in an ice bath under dark conditions, and do not store overnight to avoid affecting detection performance.
3. Sample Determination
Standard Curve Construction
Dissolve the entire 5 mg NADPH powder provided in the kit with 6 mL ultrapure water to prepare a 1 mM NADPH standard solution. Aliquot and store at −80 °C in the dark to avoid repeated freezethaw cycles. Dilute the 1 mM NADPH standard solution with NADPH diluent to final concentrations of 4 μM, 2 μM, 1 μM, 0.5 μM, 0.25 μM, 0.125 μM, 0.0625 μM, 0.03125 μM and 0 μM. The 0 μM group serves as the blank well, containing only NADPH diluent. Pipette 20 μL of each standard solution into separate wells of a 96well plate, then add 104 μL of colordeveloping working solution to each well. After mixing thoroughly, incubate at 37 °C for 10 min and measure the absorbance at A450.
Sample Determination
Pipette 20 μL of sample or diluted sample into sample wells of a 96well plate, add 104 μL of colordeveloping working solution to each well, mix well, incubate at 37 °C for 10 min, and measure the absorbance at A450.
4. Result Calculation
When establishing the standard curve, the blank well control needs to be subtracted. After the standard curve is established, the glycerol concentration (C) in the sample can be calculated according to the standard curve, and the calculation formula is as follows:
C(μM) = A ×2 n
Note: A represents the NADPH concentration (μM) calculated from the standard curve; 2n is the sample dilution factor multiplied by 2. Since an equal volume of NADP or NADPH neutralization solution is added after sample treatment, the sample is diluted by one fold correspondingly.
Precautions
1. NADPH is unstable. It is recommended to prepare the standard solution freshly before use and keep it away from light.
2. It is also recommended to prepare the chromogenic working solution freshly for immediate use.
3. NADP and NADPH extraction solutions are corrosive. Always wear a lab coat and rubber gloves in standard operating procedure to avoid skin contact.
ZJ26F1039695
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