High throughput inhibition assay using peptide based substrate. 5 μM peptide substrate, and 50 nM BoNT/A LC (black filled triangle) were used in the assay. For peptide substrate control (purple filled square), only peptide substrate was used. For EDTA inhibition (blue filled diamond), BoNT/A LC was incubated with 10 mM EDTA at 37°C for 30 min before adding substrate to the reaction wells. The assay was carried out at 37°C for 30 min. Fluorescence was read using a SpectraMax M5 fluorescence microplate reader (Molecular Devices). The excitation wavelength was 490 nm, and the emission wavelength was 523 nm, with an emission cutoff filter of 515 nm. The results shown are the average of five runs. The error bars represent plus/minus standard deviations from 5 runs.