| Assay Method Information | |
| | SPR Biosensor Assay |
| Description: | An SPR assay was used to determine the binding affinities of compounds. Experiments were performed on a Biacore 3000 (Biacore, Uppsala, Sweden) instrument with CM5 research grade chips (Biacore, Uppsala, Sweden). Sensorgrams were recorded at a frequency of 2.5 Hz. EcIspF was immobilized using amine-coupling chemistry with readings between 4500 and 5500 response units were obtained. After immobilization, a 6 min injection at 5 uL min-1 of 1 M ethanolamine was used to quench excess active succinamide ester groups. SPR binding experiments with EcIspF were performed at 10 deg C in 50 mM sodium phosphate pH 7 and 2 mM MgCl2 at a flow rate of 30 uL min-1. The sensor surface was regenerated between experiments by applying the running buffer for 20 min to dissociate any ligand complex. This was followed by a further 20 min stabilization period. The 40 min total regeneration time between experiments helped to eliminate any carry-over of EcIspF bound to ligand. Each injection at a given concentration was repeated three times. Blank injections were included for each measurement series and subtracted from the data. The equilibrium (steady state) binding curves were analyzed by nonlinear regression and fit to a one-to-one Langmuir binding model. All ligands assessed by SPR were greater than 95% purity as established by high-performance liquid chromatography or combustion analysis. |
| Affinity data for this assay | |
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