Product Introduction

This kit is a colorimetric kit based on WST-8, which detects the amount, ratio and total amount of NAD ⁺ (oxidized coenzyme I) and NADH (reduced coenzyme I) in cells, tissues or other samples by colorimetry. NAD ( Nicotinamide adenine dinucleotide ) is a coenzyme present in all cells, including two forms: NAD ⁺ ( oxidized form ) and NADH ( reduced form ) . 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. For example, deacetylases such as Sirt1 of the Sirtuins family need NAD ⁺ as a substrate for deacetylation reactions to regulate the acetylation level of proteins and thus participate in the life activities of cells. NAD ⁺ plays an important role in cells and the body. Its synthesis and degradation and its products are involved in cell apoptosis, metabolic regulation and gene expression regulation, and the reduction of NAD ⁺ is one of the main factors for cell death. The importance of NAD ⁺ in regulating the cellular redox state and its function in regulating signaling pathways and transcription make NAD ⁺ and the enzymes that synthesize and consume it potential drug targets for a variety of diseases.

Features

1.WST-8 is an upgraded substitute for MTT, and has obvious advantages over MTT or other MTT-like products such as XTT, MTS, etc. First, the formazan generated by the reduction of MTT by some dehydrogenases is not water-soluble and requires a specific dissolving solution to dissolve; while the formazan generated by WST-8, XTT, and MTS are all water-soluble, which can save the subsequent dissolution step. Secondly, the formazan generated by WST-8 is more soluble than the formazan generated by XTT and MTS. Thirdly, WST-8 is more stable than XTT and MTS, making the experimental results more stable.

2.Compared with MTT, XTT, etc., WST-8 has a wider linear range and higher sensitivity.

3.Compared with WST-1, WST-8 has higher detection sensitivity, is more soluble, and is more stable.

4.This kit is easy to use, highly sensitive, and has a wide linear range. It can detect NAD ⁺ or N ADP as low as 0.25 μM , and shows a good linear relationship between 0.25 μM and 10 μM. The detection can be performed using lysate of cells, tissues, etc., without the need to separate and purify NAD ⁺ and NADH in cells, tissues, or other samples, and can specifically detect NAD ⁺ and NADH, but not NADP ⁺ and NADPH.

Detection principle    

The traditional detection method of NAD ⁺ /NADH is to detect the change of NADH absorption wavelength at 340 nm . This method has low sensitivity and is easily interfered by similar ultraviolet absorbing substances in the sample. In addition, it is usually necessary to increase the amount of sample to compensate for the deficiency of too low absorbance of NADH at 340 nm during ultraviolet detection . Therefore, this traditional detection method has great limitations. This kit can detect the content of NAD ⁺ and NADH in the sample and their ratio. The specific principle is as follows:

a.Determination of the total amount of NAD ⁺ and NADH: Ethanol is oxidized to acetaldehyde by alcohol dehydrogenase (ADH), and NAD ⁺ is reduced to NADH in this reaction process; the generated NADH reduces WST-8 to orange-yellow formazan by the action of the electron coupling reagent 1-mPMS (1-Methoxy-5-methylphenazinium Methyl Sulfate), with a maximum absorption peak at around 450nm. The formazan generated in the reaction system is proportional to the total amount of NAD ^{+ and NADH in the sample.}

b. Determine the amount of NADH alone: After heating in a 60ºC water bath for 30 minutes, the NAD+ in the sample will decompose and only NADH will remain. NADH reduces WST-8 to formazan, and the amount of formazan generated by the reaction is determined by colorimetry, and ultimately the amount of NADH in the sample can be determined.

c. Determination of NAD ⁺ and NAD ⁺ /NADH ratio: Based on the total amount of NAD ^{+ and NADH and the amount of NADH obtained in the first two steps, the amount of NAD + in the sample} and the ratio of NAD ^{+ /NADH} can be calculated .

Product composition

Storage conditions

The unopened kit can be stored at -20℃ for 12 months.

Notes

This product is limited to scientific research by professionals and must not be used for clinical diagnosis or treatment, used as food or medicine, or stored in ordinary residences.