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Cat. No.: ENPW8-100 (for 100T)

Description

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

This product's performance and applications are essentially similar to the standard NADP+/NADPH Assay Kit (WST-8 method) available in the market, but the enhanced version of this kit features a special de-sticking and dissolving solvent added to the enhanced NADP+/NADPH extraction buffer. This addition effectively addresses the viscosity issues that may arise during the extraction of cellular and tissue samples with the standard NADP+/NADPH Assay Kit (WST-8 method), making the samples easier to aspirate and the operation more convenient.

Principle

The traditional detection method for NADP+/NADPH and NAD+/NADH involves measuring the changes in absorbance wavelength at 340nm, a method with low sensitivity that is prone to interference from UV-absorbing substances present in the samples. Additionally, compensating for the low absorbance of NADPH at 340nm typically requires larger sample volumes during UV detection, thus limiting the effectiveness of this detection method.

WST-8 is an upgraded alternative to MTT, offering distinct advantages over MTT and similar products such as XTT and MTS. Firstly, formazan generated by some dehydrogenases from MTT is not water-soluble and requires specific solutions for dissolution. However, formazan produced by WST-8, XTT, and MTS is water-soluble, eliminating the need for subsequent dissolution steps. Secondly, formazan generated by WST-8 is more easily soluble than that produced by XTT and MTS. Thirdly, WST-8 exhibits greater stability compared to XTT and MTS, resulting in more consistent experimental results. Moreover, WST-8 offers a wider linear range and higher sensitivity compared to MTT, XTT, and similar products.

In comparison to WST-1, WST-8 offers higher detection sensitivity, better solubility, and greater stability.

This assay kit offers convenient usage as it eliminates the need for the isolation and purification of NADP+ and NADPH from cells, tissues, or other samples. It specifically detects NADP+ and NADPH without detecting NAD+ and NADH. The kit can detect concentrations as low as 0.05μM (2.5pmol) of NADP+ or NADPH, exhibiting good linearity between 0.05μM (2.5pmol) and 6μM (300pmol).

NADP (Nicotinamide adenine dinucleotide phosphate) serves as a coenzyme in many redox reactions, existing in two forms: NADP+ (oxidized form) and NADPH (reduced form). NADP+ also participates in biosynthetic reactions, such as lipid and nucleic acid synthesis. In animal cells, the oxidative phase of the pentose phosphate pathway (PPP) is the primary source of NADPH.

This assay kit enables the detection of NADP+, NADPH, and their ratio in samples, based on the following principles:

a. Total measurement of NADP+/NADPH: Glucose-6-phosphate (G6P) is oxidized to 6-phosphogluconate (6-PG) by glucose-6-phosphate dehydrogenase (G6PDH), reducing NADP+ to NADPH in the process. The generated NADPH then reduces WST-8 to formazan in the presence of the electron-coupling reagent 1-mPMS (1-Methoxy-5-methylphenazinium Methyl Sulfate), exhibiting maximum absorption at around 450nm. The amount of formazan produced is proportional to the total amount of NADP+/NADPH in the sample.

b. Measurement of NADPH alone: After heating the sample in a 60°C water bath for 30 minutes, NADP+ decomposes, leaving NADPH. NADPH then reduces WST-8 to formazan, and the amount of formazan produced is determined by colorimetric methods to quantify the NADPH content in the sample.

c. Determination of NADP+ and NADP+/NADPH ratio: Based on the measurements obtained from the first two steps, the total amount of NADP+ and NADPH, as well as the amount of NADPH, can be calculated to determine the NADP+ content and NADP+/NADPH ratio in the sample.

Components

• G6PDH
• Color Development Solution
• NADPH
• Enhanced NADP+/NADH Extraction Solution
• Reaction Buffer Solution

Storage

Store at -20ºC, valid for one year. Color Development Solution and NADPH must be stored at -20ºC in the dark. After preparation, NADPH 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 frozen. Please strictly adhere to the storage conditions. If not used up at once, to avoid repeated freeze-thaw cycles leading to product degradation, please divide appropriately and store.
• NADPH is relatively unstable; after removal, please use it promptly. If the standard curve is not ideal, it is likely that the standard has degraded.
• Due to the high viscosity of the enhanced NADP+/NADPH extraction solution, when using it as a diluent, whether diluting standards or samples, it is essential to ensure uniform dilution to prevent significant fluctuations in experimental data.
• Based on testing, freezing and thawing the enhanced NADP+/NADPH extraction solution three times, and storing it at 4°C for 7 days did not significantly affect its de-sticking and dissolving activity. To ensure the stability and optimal performance of this product, after the first thaw, it is recommended to divide and store it at low temperatures to avoid repeated freeze-thaw cycles and prolonged exposure to room temperature. Repeated freeze-thaw cycles or storage at room temperature may gradually diminish the effectiveness of the de-sticking and dissolving solvent in the enhanced NADP+/NADPH extraction solution, thereby affecting the performance of the product.
• For the extraction of cellular or tissue samples, add 200μl of the enhanced NADP+/NADPH extraction solution per 100 million cells or 10mg of tissue. Typically, the enhanced NADP+/NADPH extraction solution in this product dissipates its viscous state within a few seconds after addition to the sample. However, the time for the viscosity to disappear may vary depending on factors such as the type of sample and the proportion of extraction solution added.
• During sample addition and mixing, it is essential to avoid bubble formation to prevent interference with the final absorbance measurement.
• If the reaction temperature and time cannot be strictly controlled, it is necessary to set a standard curve for each test.
• If the concentrations of NADP+ and NADPH in the sample solution are too high or too low and are outside the linear detection range of the assay kit, adjust the amount of sample or extraction solution accordingly.
• Due to the instability of NADP+ and NADPH, they are prone to degradation during freezing, so it is advisable to use fresh samples for testing whenever possible. If determining the NADP+ content and NADP+/NADPH ratio in the sample is required, it is recommended to separate the total amount of NADP+ and NADPH from the measurement of NADPH alone during the 30-minute sample heating process to decompose NADP+, thereby minimizing errors due to possible degradation during the waiting period.
• This product is intended for scientific research by professionals only and should not be used for clinical diagnosis or treatment, nor for food or drugs, and should not be stored in ordinary residential areas.
• For your safety and health, please wear lab coats and disposable gloves during operation.