| Assay Method Information | |
| | In Vitro Enzyme Assay |
| Description: | The three NOS isoforms, rat nNOS, murine iNOS and bovine eNOS were recombinant enzymes overexpressed in E. coli and purified as reported in the literature. (See, e.g., example 51 of the aforementioned incorporated '790 patent and the references cited therein.) The hemoglobin capture was used to measure nitric oxide production. (See, Hevel, J. M. and Marietta, M. A. Nitric Oxide Synthase Assays. Methods Enzymol. 1994, 133, 250-258.) Briefly, the assay was run at 37 C. in 100 mM HEPES buffer (10% glycerol; pH 7.4) in the presence of 10 uM L-arginine. The following NOS cofactors were also included in the assay: 100 uM NADPH, 10 uM tetrahydrobiopterin, 1 mM CaCl2, 11.6 ug/mL calmodulin and 3.0 uM oxyhemoglobin. For iNOS, calmodulin and CaCl2 were omitted. The assay was run in a high throughput manner, using the Synergry 4 by BioTek, at the Northwestern University HighThroughput Analysis Facility. |
| Affinity data for this assay | |
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