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

Published in Methods in Molecular Biology. 2019, vol. 1982, p. 103-111
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 assayHigh-throughput screeningMembrane purificationNOX5
PMID: 31172468
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Research group Radicaux libres et cellules souches embryonnaires (60)
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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 https://archive-ouverte.unige.ch/unige:126109

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Deposited on : 2019-11-12

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