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

Authors :
Andrew D. Napper
Sharmila Sivendran
Source :
Current Protocols in Chemical Biology
Publication Year :
2014

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.

Details

ISSN :
21604762
Volume :
3
Issue :
2
Database :
OpenAIRE
Journal :
Current protocols in chemical biology
Accession number :
edsair.doi.dedup.....733984e60a99bf180ba5a3f94b47fc2e