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Cat. No.: ENW8-100 (for 100T)

Description

Enhanced NAD+/NADH Assay Kit with WST-8 is a colorimetric assay based on the WST-8 reaction, used for the quantitative determination of NAD+ (oxidized form of nicotinamide adenine dinucleotide) and NADH (reduced form of nicotinamide adenine dinucleotide), as well as their ratio and total amounts in cells, tissues, or other samples.

While the performance and applications of this product are essentially similar to the standard NAD+/NADH Assay Kit (WST-8 method) available in the market, it contains a special de-sticking and dissolving solvent in the NAD+/NADH extraction buffer. This addition effectively addresses the viscosity issues that may arise during the extraction of cellular and tissue samples with the standard NAD+/NADH Assay Kit, making the samples easier to aspirate and the operation more convenient. Additionally, it significantly improves the extraction efficiency of cellular and tissue samples, thereby enhancing the detection sensitivity of the samples. This enhancement allows achieving equivalent results to the standard NAD+/NADH Assay Kit with either a reduced sample volume or shorter incubation time. For the same volume of sample prepared with both extraction buffers, the signal intensity of the enhanced kit is 20%-60% higher than that of the standard kit.

Principle

Traditional detection methods for NAD+/NADH and NADP+/NADPH involve measuring the absorbance changes of NADH or NADPH at 340nm. However, this method has low sensitivity and is prone to interference from similar UV-absorbing substances in samples. Additionally, in UV detection, larger sample volumes are often required to compensate for the low absorbance of NADH at 340nm, thus limiting the method's applicability.

WST-8 is an upgraded alternative to MTT with distinct advantages over MTT or other similar products like XTT and MTS. Firstly, the formazan produced by some dehydrogenases from MTT is insoluble in water and requires specific solutions for dissolution, whereas the formazan generated by WST-8, XTT, and MTS is water-soluble, eliminating the need for subsequent dissolution steps. Secondly, the formazan produced by WST-8 is more soluble than that produced by XTT and MTS. Thirdly, WST-8 exhibits greater stability compared to XTT and MTS, ensuring more stable experimental results. Moreover, WST-8 offers a wider linear range and higher sensitivity compared to MTT, XTT, and similar products.

Compared to WST-1, WST-8 offers higher detection sensitivity, greater solubility, and increased stability.

This assay kit is convenient to use, as it does not require the separation or purification of NAD+ and NADH from cells, tissues, or other samples. It can specifically detect NAD+ and NADH without detecting NADP+ and NADPH. The kit can detect NAD+ or NADH levels as low as 0.25μM (5pmol) and shows good linearity between 0.25μM (5pmol) and 10μM (200pmol).

NAD (Nicotinamide adenine dinucleotide) is a coenzyme present in all cells, existing in two forms: NAD+ (oxidized form) and NADH (reduced form). NAD+ serves as a coenzyme in redox reactions, as well as a substrate for many enzymes involved in cellular reactions. For example, Sirtuins such as Sirt1 require NAD+ as a substrate for deacetylation reactions, regulating protein acetylation levels and participating in cellular processes. NAD+ plays crucial roles in cellular and physiological functions, including apoptosis, metabolic regulation, and gene expression control. Decreased levels of NAD+ are considered a major factor in cellular death. Although NMNAT (nicotinamide mononucleotide adenylyltransferase) is a synthetic enzyme for NAD+, including Nmnat1, Nmnat2, and Nmnat3, NAMPT (Nicotinamide phosphoribosyltransferase) is generally considered the rate-limiting enzyme for NAD+ synthesis. The importance of NAD+ in regulating cellular redox status and its functions in signaling pathways and transcription make NAD+ and its synthetic and degradative enzymes potential targets for various diseases.

This assay kit can detect the levels of NAD+, NADH, and their ratio in samples. The principles are as follows:

a. Total amount determination of NAD+ and NADH: Ethanol is oxidized to acetaldehyde by alcohol dehydrogenase (ADH), reducing NAD+ to NADH. The generated NADH reduces WST-8 to formazan, which exhibits maximal absorption at around 450nm. The amount of formazan produced is proportional to the total amount of NAD+ and NADH in the sample.

b. Determination of NADH alone: After heating at 60°C for 30 minutes, NAD+ decomposes, leaving only NADH. NADH reduces WST-8 to formazan, and the amount of formazan produced is determined by colorimetric assay, allowing for the quantification of NADH in the sample.

c. Determination of NAD+ and NAD+/NADH ratio: Based on the total amounts of NAD+ and NADH obtained from the previous steps, along with the amount of NADH determined separately, the quantity of NAD+ and the NAD+/NADH ratio in the sample can be calculated.

Components

• Ethanol Dehydrogenase
• Color Development Solution
• NADH
• NADH Preparation Solution
• Enhanced NAD+/NADH Extraction Solution
• Reaction Buffer Solution

Storage

Store at -20ºC, valid for one year. Color Development Solution and NADH must be stored at -20ºC in the dark. After preparation, NADH should be aliquoted appropriately and stored at -80ºC. Avoid repeated freeze-thaw cycles for all reagents.

Precautions

• All reagents in this assay kit need to be stored under refrigeration. Please strictly adhere to the storage conditions. If not used in a single operation, to avoid product degradation due to repeated freeze-thaw cycles, please aliquot appropriately before storage.
• NADH is relatively unstable, so please use it as soon as it's taken out. If you observe unsatisfactory standard curves, it's likely that the standard has degraded.
• Due to the viscosity of the enhanced NAD+/NADH extraction solution, when using it as a diluent, whether for standards or samples, it's crucial to ensure uniform dilution during the process to prevent significant fluctuations in experimental data.
• Tests have shown that freezing and thawing the enhanced NAD+/NADH extraction solution three times, storing it at 4°C for 7 days, has no significant impact on its de-sticking and dissolving activity. However, to ensure the stability and effectiveness of this product, after the first thaw, appropriate aliquoting and storage at low temperatures are recommended to avoid repeated freeze-thaw cycles and prolonged exposure to room temperature. Repeated freeze-thaw cycles or prolonged storage at room temperature may lead to a gradual loss of efficacy of the de-sticking and dissolving solvent in the enhanced NAD+/NADH extraction solution, thereby affecting the performance of this product.
• For the extraction of cellular or tissue samples, add the enhanced NAD+/NADH extraction solution at a ratio of 200μl per 100 million cells or 10mg of tissue. Typically, the enhanced NAD+/NADH extraction solution in this product will lose its viscous state within a few seconds after adding it to the sample. However, the time for the viscous state to disappear may vary depending on experimental conditions such as sample type and the proportion of extraction solution added.
• During sample addition and mixing, try to avoid bubble formation to prevent interference with the final absorbance measurements.
• If it's not possible to strictly control reaction temperature and time, setting a standard curve is necessary for each detection.
• If the concentrations of NAD+ and NADH in the sample solution are too high or too low and fall outside the linear detection range of the kit, adjust the amount of sample or extraction solution accordingly.
• Due to the instability of NAD+ and NADH, they are prone to degradation during freezing, so it's advisable to use fresh samples for testing whenever possible. If NAD+ and the NAD+/NADH ratio in the sample need to be determined, separate the measurement of the total amount of NAD+ and NADH from the measurement of NADH alone. Try to minimize errors caused by possible degradation during waiting by performing the measurements separately.
• This product is intended for scientific research by professionals only and should not be used for clinical diagnosis or treatment, nor for food or drug purposes. Do not store it in residential areas.
• For your safety and health, wear laboratory attire and disposable gloves during operation.