NOX5 Cell-Free Assay for the High-Throughput Screening of Small Molecules
| Autor: | Delphine Rasti, Yves Cambet, Vincent Jaquet, Fiona Augsburger |
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| Rok vydání: | 2019 |
| Předmět: |
0301 basic medicine
Gene isoform chemistry.chemical_classification Phagocyte Chemistry High-throughput screening 030204 cardiovascular system & hematology Small molecule Transmembrane protein 03 medical and health sciences 030104 developmental biology 0302 clinical medicine Membrane medicine.anatomical_structure Enzyme Biochemistry cardiovascular system medicine NOx circulatory and respiratory physiology |
| Zdroj: | Methods in Molecular Biology ISBN: 9781493994236 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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