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