Related Experiment Video
Updated: Apr 19, 2026
A Semi-High-Throughput Adaptation of the NADH-Coupled ATPase Assay for Screening Small Molecule Inhibitors
Published on: August 17, 2019
Miniaturized High-Throughput Fluorescent Assay for Conversion of NAD(P)H to NAD(P)
Andrew D Napper1, Sharmila Sivendran1
1Penn Center for Molecular Discovery, Institute for Medicine and Engineering, and Department of Chemical and Biomolecular Engineering, University of Pennsylvania, 1160 Vagelos Research Laboratories, 3340 Smith Walk, Philadelphia, PA 19104.
Abstract:
A miniaturized fluorescent assay is described that monitors the conversion of NADPH to NADP+. The same assay format may also be used to measure NADH to NAD+ conversion. Examples of assay development and validation results are presented to illustrate the use of this protocol to screen an enzyme that consumes NADPH as a cofactor during conversion of substrate to a reduced product. Enzymatic assays are carried out low volume 384-well plates, in which the turnover of NADPH is monitored by the decrease in fluorescent emission at 460 nm between an initial fluorescence measurement and a second reading after 90 minutes. A follow-up assay is used to rule out false positive artifacts arising from compounds that fluoresce at 460 nm.
