Miniaturized High-Throughput Fluorescent Assay for Conversion of NAD(P)H to NAD(P)
Published Online: 1 JUN 2011
Copyright © 2011 by John Wiley and Sons, Inc.
Lab Protocol Title
Current Protocols in Chemical Biology
How to Cite
Napper, A. D. and Sivendran, S. 2011. Miniaturized High-Throughput Fluorescent Assay for Conversion of NAD(P)H to NAD(P). Current Protocols in Chemical Biology. 3:81–97.
- Published Online: 1 JUN 2011
- Published Print: JUN 2011
This unit describes a miniaturized fluorescence assay 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 in low-volume 384-well plates, in which the turnover of NADPH is monitored by the decrease in fluorescence emission at 460 nm between an initial measurement and a second reading after 90 min. A follow-up assay is used to rule out false-positive artifacts arising from compounds that fluoresce at 460 nm. Curr. Protoc. Chem. Biol. 3:81-97 © 2011 by John Wiley & Sons, Inc.
- NADPH detection;
- NADH detection;
- high-throughput screening;
- enzyme assay;
- fluorescent artifact