Nicotinamide Adenine Dinucleotide (NAD) is a co-enzyme indispensible for the activity of multiple catalytic proteins. NAD+ and NADH, its reduced form, participate in many biological redox reactions. NAD+ is mostly associated with redox enzymes required for catabolic reaction and energy production. Assay Genie's NADH/NAD Quantification Kit provides a convenient tool for a sensitive detection of NADH, NAD and their ratio. There is no requirement to purify NADH or NAD from samples. The NADH selective developer develops color while reacting with NADH and can be detected at OD 450 nm. The total NAD can be easily quantified by comparing with the provided NADH standard. The method is quantitative, rapid, simple, sensitive, and designed for high throughput format. The kit can detect as low as 0.04 pmol of NADH in a 384 well assay plate. The reaction specifically detects NADH and NAD, but not NADP nor NADPH.

Figure: a) Standard Curve for NADH, NAD and heated NADH (Ht-NADH) and NAD (Ht-NAD); both heated 30 min at 60 °C; (b) Quantitation of NADH in freshly processed rat liver and kidney tissue. Tissue homogenates were prepared following kit’s protocol and deproteinized by 10 kDa spin column (12000 x g, 10 min, 4°C). 5 µl filtrates were assayed according to the kit’s protocol. (c) Quantitation of NADH in human serum and urine. Samples were centrifuged and supernatant was collected and deproteinized by 10 kDa spin column. Filtrate was spiked with 10 pmol of NADH Standard, assayed following kit’s protocol. All standards and samples absorbance readings were taken after 60 minutes cycling reaction at room temperature.