NAD/NADH-Glo™ Assay

Description

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.

Features – Benefits

• High Sensitivity: High sensitivity of the assay enables detection of total NAD⁺ and NADH directly in the wells. Fewer cells are required, with no sample preparation.
• Homogeneous, One-Step Protocol: Total NAD⁺ and NADH is measured directly in wells of a 96- or 384-well cell culture plate with one reagent addition. A simple in-plate protocol is provided for individual NAD⁺ and NADH measurements.
• Large Assay Window: The NAD/NADH-Glo™ Assay detects 10nM to 400nM NAD⁺ or NADH. The assay detects 100nM with a signal higher than fivefold over background and an assay window (maximum signal-to-background ratio) of ≥100.
• Automation Compatible: The assay is compatible with automated and high-throughput protocols. Reactions are scalable and can be performed at low volumes in 96-, 384- and 1536-well plates.
• Reliability and Reproducibility: The NAD/NADH-Glo™ Assay routinely yields Z´ factors >0.7.
• Luminescence-Based NAD⁺ and NADH Detection: The luminescent format avoids fluorescent interference due to reagents and test compounds sometimes seen in fluorescent assays.

Applications

• Monitoring changes in cellular levels of total NAD⁺ and NADH.
• Determining NAD/NADH ratios.
• Monitoring the effects of small molecule compounds on NAD⁺ and NADH levels in enzymatic reactions or directly in cells in high-throughput formats.

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