Unit

Miniaturized High-Throughput Fluorescent Assay for Conversion of NAD(P)H to NAD(P)

  1. Andrew D. Napper1,2,
  2. Sharmila Sivendran1,3

Published Online: 1 JUN 2011

DOI: 10.1002/9780470559277.ch100155

Current Protocols in Chemical Biology

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.

Author Information

  1. 1

    Penn Center for Molecular Discovery, Institute for Medicine and Engineering and Department of Chemical and Biomolecular Engineering, University of Pennsylvania, Philadelphia, Pennsylvania

  2. 2

    Nemours Center for Childhood Cancer Research, Wilmington, Delaware

  3. 3

    GlaxoSmithKline, Collegeville, Pennsylvania

Publication History

  1. Published Online: 1 JUN 2011
  2. Published Print: JUN 2011

Abstract

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.

Keywords:

  • NADPH detection;
  • NADH detection;
  • fluorescence;
  • high-throughput screening;
  • oxidoreductase;
  • enzyme assay;
  • fluorescent artifact