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The NAD/NADH-Glo Assay is a bioluminescent, homogeneous single-reagent-addition assay to detect total oxidized and reduced nicotinamide adenine dinucleotides (NAD+ and NADH, respectively) and determine their ratio in biological samples or in defined enzyme reactions. NAD Cycling Enzyme is used to convert NAD+ to NADH. In the presence of NADH, the provided reductase enzyme reduces a proluciferin reductase substrate to form luciferin, which is quantified using Ultra-Glo Recombinant Luciferase; the light signal produced is proportional to the amount of NAD+ and NADH in the sample. Cycling between NAD+ and NADH by NAD Cycling Enzyme and Reductase increases assay sensitivity and provides selectivity for the nonphosphorylated NAD+ and NADH compared to the phosphorylated forms NADP+ and NADPH. The simple add-mix-read protocol and scalable assay chemistry make the NAD/NADH-Glo Assay well suited to monitor effects of small molecule compounds on NAD and NADH levels in high-throughput formats.
