NAD/NADH Assay Kit II (colorimetric)

Product details

NAD/NADH Assay Kit II (Colorimetric) (ab221821) provides a sensitive and robust method to measure NAD⁺, NADH and their ratio in mammalian cell lysates. The assay is based on an ADH and diaphorase coupled-reaction that convers WST-1 to WST-1 formazan, which can be easily detected at OD 450 nm. As the reaction is not stopped, it is necessary to monitor the absorbance increase of WST-1 formazan at regular intervals after the reaction is initiated to determine the reaction velocity.

This assay requires purification of NAD⁺ and NADH from the cell lysates, which raises the efficiency of the reaction and increases the detection sensitivity.

Nicotinamide nucleotides are key players in the energy and oxidation-reduction reactions of a cells. Nicotinamide adenine dinucleotide (NAD) exists in two forms, an oxidized form, NAD+, and a reduced form, NADH. NAD functions as a cofactor in the vast majority of cellular redox reactions, carrying reducing equivalents from one reaction to another. Therefore, maintaining appropriate levels of NAD is essential for maintaining normal cellular respiratory function. There are two major pathways in NAD biosynthesis. The de novo pathway is maintained by the rate-limiting enzyme nicotinamide phosphoribosyltransferase (NAMPT), whereas the savage pathway recycles degraded NAD products such as nicotinamide. Studies have shown that cytosolic NAD⁺ concentrations range from 300 nM in mammalian cells to 2 mM in yeast. Depletion of NAD in cells is a major cause of cell death.

The importance of NAD function in modulating cellular redox status and controlling signaling and transcriptional events makes NAD an important cofactor when investigating normal cellular function.