The NAD/NADH-Glo Assay is a bioluminescent, homogeneous single-reagent-addition assay for detecting total oxidized and reduced nicotinamide adenine dinucleotides and determining their ratio in biological samples or in defined enzyme reactions.

• Bioluminescent Assays to Detect NAD(P)+ and NAD(P)H Levels in Cells
• Homogeneous, single-reagent-addition assays
• Easily adapted for high throughput screening formats

The NAD/NADH-Glo Assay is a bioluminescent, homogeneous single-reagent-addition assay for detecting total oxidized and reduced nicotinamide adenine dinucleotides (NAD+ and NADH, respectively) and determining their ratio in biological samples or in defined enzyme reactions. An 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. Luciferin then is quantified using Ultra-Glo Recombinant Luciferase, and the light signal produced is proportional to the amount of NAD+ and NADH in the sample. Cycling between NAD+ and NADH by the 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 NAD Cycling Enzyme, Reductase and luciferase reactions are initiated by adding an equal volume of NAD/NADH-Glo Detection Reagent, which contains NAD Cycling Enzyme and Substrate, Reductase, Reductase Substrate and Ultra-Glo Recombinant Luciferase, to an NAD+- or NADH-containing sample. Detergent present in the reagent lyses cells, allowing detection of total cellular NAD+ and NADH in a multiwell format with addition of a single reagent. An accessory protocol is provided to allow separate measurements of NAD+ and NADH, and calculation of the NAD+ to NADH ratio. 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.

The NADP Cycling Enzyme, Reductase, and luciferase reactions are initiated by adding an equal volume of NADP/NADPH-Glo Detection Reagent, which contains NADP cycling enzyme and substrate, reductase, pro-luciferin reductase substrate, and Ultra-Glo Recombinant Luciferase, to an NADP+- or NADPH-containing sample. Detergent present in the reagent lyses cells, allowing detection of total cellular NADP+ and NADPH in a multiwell format with addition of a single reagent. The one-step protocol is useful for screening changes in total NADP+ and NADPH levels. An accessory protocol is provided to allow separate measurements of NADP+ and NADPH and calculation of the NADP+ to NADPH ratio. The simple add-mix-read protocol and scalable assay chemistry make the NADP/NADPH-Glo Assay well suited to monitor effects of small-molecule compounds on NADP and NADPH levels in high-throughput formats.

In the presence of NADPH, a reductase enzyme reduces a pro-luciferin reductase substrate to form luciferin. Luciferin is then quantified using Ultra-Glo™ Recombinant Luciferase, and the light signal produced is proportional to the amount of NADP+ and NADPH in the sample. Cycling between NADP+ and NADPH by the NADP cycling enzyme and reductase increases assay sensitivity and provides selectivity for the phosphorylated NADP+ and NADPH compared to the nonphosphorylated forms NAD+ and NADH.