| Assay Method Information | |
| | SidA Activity Assay |
| Description: | Briefly, this assay consists of the formation of nitrite from hydroxyl amine that is quantified by formation of a chromophore with α-naphthylamine, which has an absorbance maximum at 529 nm. Formation of HO-Orn was quantified using a standard curve with hydroxylamine (25−350 μM). The standard assay contained 25 μL of 100 mM sodium phosphate (pH 7.5), 1 μM SidA, 500 μM L-Orn, and 250 μM NADPH for activity and IC50 determinations. Compound concentration was varied from 0.01 to 1 mM, and the reaction mix contained a final DMSO concentration of 10%. The reaction was started with the addition of NADPH, allowed to proceed for 20 min atRT, and quenched with 13 μL of 2 N HClO4. To identify compounds that caused inhibition by inducing protein aggregation, the IC50 was determined in 100 mM phosphate buffer, pH 7.5, containing 0.1% v/v Triton X-100.23 As some compounds had either an increase or decrease in the color development or had absorbance at a visi |
| Affinity data for this assay | |
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