NAD/NADH Quantitation Kit

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 preparationCell 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, discardClear, 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. Specification: sufficient for 100colorimetrictests Related Documents: https://ald-pub-files.oss-cn-shanghai.aliyuncs.com/aladdinsci/pdp/sds/1/N486176-SCI_eb9f97ba3006c82b9afc36977f20a88c.pdf