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Scientific article
English

NOX5 cell-free assay for the high-throughput screening of small molecules

Published inMethods in Molecular Biology, vol. 1982, p. 103-111
Publication date2019
Abstract

NADPH oxidases (NOX) are a family of transmembrane enzymes, which catalyze the formation of O2˙- and H2O2. Membrane fractions of leukocytes are highly enriched in the phagocyte NOX isoform (NOX2). This feat has allowed the development of a complex NOX2 cell-free assay, which has been a key tool for the understanding of the mode of action of NOX2, its biochemistry, pharmacology, and identification of NOX2-specific inhibitors. In addition to NOX2, there are six other NOX isoforms in humans, but cell-free assays of non-phagocytic oxidases are infrequently used, and their specificity has recently been debated. Here we describe a NOX5 cell-free assay. We present a method to purify the membranous component of cells stably transduced with NOX5 and to measure O2˙- in a high-throughput format (96-w or 384-w plates). The experimental description allows high-throughput screening of small molecules with limited cost.

Keywords
  • Cell-free assay
  • High-throughput screening
  • Membrane purification
  • NOX5
Citation (ISO format)
AUGSBURGER, Fiona et al. NOX5 cell-free assay for the high-throughput screening of small molecules. In: Methods in Molecular Biology, 2019, vol. 1982, p. 103–111. doi: 10.1007/978-1-4939-9424-3_6
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ISSN of the journal1064-3745
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