The NADP/NADPH-Glo Assay is a bioluminescent, homogeneous, single-reagent-addition method for rapid detection of total oxidized and reduced nicotinamide adenine dinucleotide phosphates (NADP+ and NADPH, respectively) and determining their ratio in biological samples and defined enzyme reactions. An NADP cycling enzyme is used to convert NADP+ to NADPH. In the presence of NADPH, a 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 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. 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, proluciferin 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.|
High Sensitivity: High sensitivity of the assay enables detection of total NADP+ and NADPH directly in the wells. Fewer cells are required, with no sample preparation. Homogeneous, One-Step Protocol: Total NADP+ and NADPH 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 NADP+ and NADPH measurements. Large Assay Window: The NADP/NADPH-Glo(TM); Assay detects 10nM to 400nM NADP+ or NADPH. 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 NADP/NADPH-Glo(TM); Assay routinely yields Z factors more than0.7. Luminescence-Based NADP+ and NADPH Detection: The luminescent format avoids fluorescent interference due to reagents and test compounds sometimes seen in fluorescent assays.