NAD/NADH-Glo™ and NADP/NADPH-Glo™ Assays

Specificity is imparted to the NAD/NADH-Glo™ and NADP/NADPH-Glo™ Assay reactions by including NAD or NADP Cycling Enzyme and NAD or NADP Cycling Substrate to selectively measure nonphosphorylated and phosphorylated nucleotides, respectively. Accessory protocols are provided to allow separate measurements of NAD+ and NADH or NADP+ and NADPH and calculation of the ratio of the oxidized and reduced forms.

NADH, NADPH, NAD+ and NADP+ were prepared and diluted to the indicated concentrations in phosphate-buffered saline.

The simple add-and-read protocols are compatible with automated and high-throughput workflow, and the flexible chemistries adapt to 96-, 384- and 1,536-well plate formats and high-throughput systems. They yield Z′ values >0.7. Assays were performed using 8μl of the indicated concentration of NADH or NADPH with 8μl of the appropriate Luciferin Detection Reagent in 384-well plates. S/B = signal-to-background ratio.