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Assay Method Information |
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| | Steady-State Kinetic Assays |
| Description: | Enzymes were diluted to 10 nM in PBS pH 6.5 (adjusted with sodium acetate) supplemented with 0.1 g/L pluronic F127 (Sigma). Varying concentrations of resorufin acetate (Sigma) in DMSO were added as 5 μL aliquots to clear-bottom 96-well plates (Greiner bio-one). Using a multi-channel pipette, 95 μL of enzyme was added to each well and quickly mixed by pipetting up and down several times. Each enzyme was assayed in 2 separate runs, each with 4 replicates per substrate concentration and 4 replicates of catalytic-dead enzyme. Fluorescence was measured every 35 seconds for 15 minutes on a Tecan F500 plate reader (525/35 nm excitation filter, 600/10 nm emission filter, and a 560 LP dichroic filter). |
| Affinity data for this assay | |
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