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NADPH Oxidases pp 103-111 | Cite as

NOX5 Cell-Free Assay for the High-Throughput Screening of Small Molecules

  • Fiona Augsburger
  • Delphine Rasti
  • Yves Cambet
  • Vincent JaquetEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1982)

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.

Key words

NOX5 Membrane purification Cell-free assay High-throughput screening 

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Fiona Augsburger
    • 1
  • Delphine Rasti
    • 1
  • Yves Cambet
    • 1
    • 2
  • Vincent Jaquet
    • 1
    • 2
    Email author
  1. 1.Department of Pathology and Immunology, Faculty of MedicineUniversity of GenevaGenevaSwitzerland
  2. 2.READS Unit, Faculty of MedicineUniversity of GenevaGenevaSwitzerland

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