J2513801计算溶液所需的质量、体积或浓度。
为什么选择此级别
，适用于对基线干扰要求严格的色谱和分析工作流程。
储存与运输
-20°C储存。超低温运输 。请查阅批次 COA 获取详细规格。
质量文档
SDS、COA、产品数据表及规格说明书均可下载。可通过批号查询获取批次 COA。
文献证明
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。
概述
烟酰胺腺嘌呤二核苷酸(Nicotinamide adenine dinucleotide, NAD)是所有细胞中都存在的一种辅酶，包括NAD+(氧化型)和NADH(还原型)两种形式。NAD+既是氧化还原反应过程中传递电子的辅酶，又可以作为很多酶的底物来参与细胞内反应。NAD+在细胞和体内发挥着重要的功能，其合成和降解及其产物参与细胞凋亡、代谢调控和基因表达的调控等，并且NAD+的减少是细胞死亡的主要因素之一。NAD+在调节细胞氧化还原状态方面的重要性以及调控信号通路及转录方面的功能，使得NAD+及其合成和消耗的酶成为多种疾病的潜在药物靶点。传统的NAD+/NADH和NADP+/NADPH测定是通过检测340 nm处的吸收来完成的，该方法灵敏度低且易受干扰。NAD/NADH检测试剂盒是一种基于WST-8的显色反应，通过比色法来检测细胞、组织或其它样品中NAD+(氧化型辅酶Ⅰ)和NADH(还原型辅酶Ⅰ)各自的量、比值和总量的检测试剂盒。NAD/NADH检测试剂盒能特异性地检测NAD+和NADH，而不检测NADP+和NADPH，在反应过程中NAD+被还原为NADH，NADH将WST-8还原成橙黄色formazan（甲臜），在450 nm左右有最大吸收峰。反应体系中生成的formazan与样品中NAD+或NADH的总量呈比例关系。本试剂盒可检测0.1 μM-10 μM NAD+或NADH。
组分：
A. NAD+/NADH提取液；B. 乙醇脱氢酶；C. 显色液；D. NADH；E. 10× NADH配制液；F. 反应缓冲液；
注意事项：
1. 建议NADH标准品溶解后小量分装保存，避免反复冻融；
2. NAD+/NADH提取液粘稠，使用过程中务必保证和待加入的体系充分混匀；
3. 加样和混匀过程中，应避免产生气泡影响最终的吸光度检测；
4. 由于NAD+/NADH不稳定，易降解，所以尽量使用新鲜样品进行检测；
5. 为了您的安全和健康，请穿实验服并戴一次性手套操作。
使用方法:
1. 样品准备
细胞样品（贴壁细胞或悬浮细胞）：收集约 1×106 个细胞，离心去除培养液，用预冷的 PBS 清洗细胞，离心 5 min，弃上清，加入 200 μL 冰浴预冷的 NAD+/NADH 提取液，轻轻吹打以促进细胞的裂解，裂解过程在室温或冰上操作均可。裂解结束后 12,000 g，4ºC 离心 5-10 min，取上清备用。组织样品：冰上预冷的 PBS 清洗组织后，称取样品 10-30 mg，用剪刀剪碎，置于匀浆器中，加入 400 μL NAD+/NADH 提取液于室温下或冰上进行匀浆，结束后 12,000 g，4℃离心 5-10 min，取上清备用。
2. 实验准备
（1）配制 10 mM NADH 标品：10×NADH 配制液先用水稀释为 1×的 NADH 配制液，再吸取 141.0 μL（20T）/704.8 μL（100T）1× NADH 配制液加入 D 组分所在的管中，充分溶解，得到 10 mM NADH 标品溶液，适当分装后置于-80℃冰箱，避光保存。
（2）稀释标品：将 10 mM 的 NADH 标品用 NADH 配制液稀释成合适的浓度梯度，如 0、0.25、0.5、1、2、4、6、8、10 μM，向 96 孔板中每孔加入 20 μL 的标准品。如有需要，可根据样品中 NADH 的含量适当调整标品浓度范围。
（3）乙醇脱氢酶工作液的配制（现用现配）：用反应缓冲液将乙醇脱氢酶稀释 45 倍。检测时，向每个标品或样品中加入 90 μL 乙醇脱氢酶工作液。
3. 样品测定
（1）NAD+/NADH 总量测定：吸取 20 μL 待测样品于 96 孔板中，设置重复。如样品中的 NAD+或 NADH 含量过高，超出标准曲线的范围，则需用 NADH 配制液适当稀释后再进行检测，含量过低时应增加样品的用量。
（2）样品中 NAD+、NADH 含量或 NAD+/NADH 比值的测定：向离心管中加入 50-100 μL 待测样品，60℃加热 30 min 以分解 NAD+，如果加热后产生不溶物，10000 g，室温或 4℃离心 5 min，吸取 20 μL 上清至 96 孔板中待测，设置重复。如样品中的 NAD+或 NADH 含量过高，超出标准曲线的范围，则需用 NADH 配制液适当稀释后再进行检测，含量过低时应增加样品的用量。
（3）在 96 孔板中按照如下方式加样：
（4）每孔加入 10 μL 显色液，充分混匀后，室温孵育 30 min，测 450 nm 处的吸光度。
（5）数据分析。根据标准曲线计算样品中的 NAD+和 NADH 总浓度或 NADH 的浓度，通过样品加入的体积即可计算出 NAD+、NADH、NAD+/NADH 总量。
Nicotinamide adenine dinucleotide ( NAD ) is a coenzyme present in all cells, including NAD + ( oxidized ) and NADH ( reduced ) forms. NAD + is not only a coenzyme that transfers electrons during redox reactions, but also can be used as a substrate for many enzymes to participate in intracellular reactions. NAD + plays an important role in cells and in vivo. Its synthesis and degradation and its products are involved in apoptosis, metabolic regulation and gene expression regulation, and the reduction of NAD + is one of the main factors of cell death. The importance of NAD + in regulating the redox state of cells and the function of regulating signaling pathways and transcription make NAD + and the enzymes it synthesizes and consumes become potential drug targets for many diseases.The traditional determination of NAD + / NADH and NADP + / NADPH is accomplished by detecting the absorption at 340 nm. This method has low sensitivity and is susceptible to interference. NAD / NADH detection kit is a color reaction based on WST-8, by colorimetric method to detect cells, tissues or other samples of NAD + ( oxidized coenzyme I ) and NADH ( reduced coenzyme I ) respective amount, ratio and total amount of detection kit. The NAD / NADH detection kit can specifically detect NAD + and NADH, but not NADP + and NADPH. During the reaction, NAD + is reduced to NADH, and NADH reduces WST-8 to orange formazan, with a maximum absorption peak at about 450 nm. The formazan formed in the reaction system was proportional to the total amount of NAD + or NADH in the sample. The kit can detect 0.1 μM-10 μM NAD + or NADH.
Composition:
A. NAD+/NADH extraction solution; B. Ethanol dehydrogenase; C. Color developing solution; D. NADH; E. 10 × NADH preparation solution; F. Reaction buffer;
Matters needing attention：
1.It is recommended that the NADH standard should be dissolved and stored in small quantities to avoid repeated freezing and thawing ;
2.the NAD + / NADH extract is sticky, and it is necessary to ensure that it is fully mixed with the system to be added during use ;
3.In the process of sample addition and mixing, bubbles should be avoided to affect the final absorbance detection ;
4.Because NAD + / NADH is unstable and easy to degrade, so try to use fresh samples for detection ;
5.For your safety and health, please wear experimental clothes and disposable gloves.
Usage:
1. Sample preparation
Cell samples (adherent cells or suspended cells): Collect approximately 1 × 106 cells, centrifuge to remove culture medium, wash cells with pre cooled PBS, centrifuge for 5 minutes, discard
Clear, add 200 μ L ice bath pre cooled NAD+/NADH extract, gently blow to promote cell lysis, and the lysis process can be operated at room temperature or on ice. After cracking, 12000 g was centrifuged at 4 º C for 5-10 minutes, and the supernatant was taken for later use.
Organizational sample: After cleaning the tissue with pre cooled PBS on ice, weigh 10-30 mg of the sample, cut it into small pieces with scissors, place it in a homogenizer, and add 400 μ L NAD+
/The NADH extraction solution was homogenized at room temperature or on ice. After completion, 12000 g was centrifuged at 4 ℃ for 5-10 minutes, and the supernatant was taken for later use.
2. Experimental preparation
(1) Prepare 10 mM NADH standard: 10 × Dilute NADH preparation solution with water to 1 first × Prepare NADH solution and then aspirate 141.0 μ L (20T)/704.8 μ L (100T) 1 × Add NADH preparation solution to the tube containing component D, dissolve thoroughly, and obtain a 10 mM NADH standard solution. After appropriate packaging, store it in a -80 ℃ refrigerator in the dark.
(2) Dilution standard: Dilute 10 mM NADH standard with NADH preparation solution to an appropriate concentration gradient, such as 0, 0.25, 0.5, 1, 2, 4, 6, 8, 10 μ M. Add 20 to each well of the 96 well plate μ Standard product of L. If necessary, the standard concentration range can be adjusted appropriately based on the NADH content in the sample.
(3) Preparation of ethanol dehydrogenase working solution (currently used and prepared): Dilute ethanol dehydrogenase 45 times with reaction buffer. During testing, add 90% to each standard or sample μ L ethanol dehydrogenase working solution.
3. Sample determination
(1) Total NAD+/NADH determination: Take 20% μ Place the sample to be tested in a 96 well plate and repeat the setup. If the NAD+or NADH content in the sample is too high and exceeds the range of the standard curve, it is necessary to dilute it appropriately with NADH preparation solution before testing. If the content is too low, the amount of sample should be increased.
(2) Determination of NAD+, NADH content or NAD+/NADH ratio in the sample: Add 50-100 to the centrifuge tube μ L sample to be tested, heated at 60 ℃ for 30 minutes to decompose NAD+. If an insoluble substance is produced after heating, 10000 g is centrifuged at room temperature or 4 ℃ for 5 minutes, and 20% is taken μ L is cleaned and placed in a 96 well plate for testing, with repeated settings. If the NAD+or NADH content in the sample is too high and exceeds the range of the standard curve, it is necessary to dilute it appropriately with NADH preparation solution before testing. If the content is too low, the amount of sample should be increased.
(3) Add samples in a 96 well plate as follows:
(4) Add 10 to each hole μ L color solution, thoroughly mix, incubate at room temperature for 30 minutes, and measure the absorbance at 450 nm.
(5) Data analysis. Calculate the total concentration of NAD+and NADH or the concentration of NADH in the sample based on the standard curve. The total amount of NAD+, NADH, and NAD+/NADH can be calculated by the volume of the sample added.
规格
英文别名
NAD/NADH Assay Kit
规格或纯度
足以进行100次比色测试
英文名称
NAD/NADH Quantitation Kit
储存与运输
储存条件
-20°C储存
运输条件
超低温运输
技术文档
高级数据
质检证书（CoA,COO,BSE/TSE 和分析图谱)
技术文档和文章
溶液计算器
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