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| | SPR Binding Affinity Assay (Standard) |
| Description: | Standard kinetics: an independent association-dissociation cycle was run for each concentration by injecting compound solution and waiting for dissociation before the injection of the following concentration. SPR experiments were performed at 25 °C using a Biacore T200 instrument (GE Healthcare). PBS (pH 7.4) supplemented with 0.05% Tween 20 was used as running buffer. Human FD and FB were immobilized covalently to a Series S Sensor Chip CM5 (GE Healthcare) at a flow rate of 10 μL/min using an amine-coupling protocol. Reagents for the immobilization were purchased from GE Healthcare (Amine Coupling Kit; BR-1000-50). The sensor-chip surface was activated by a 5 min injection of a 1:1 (v/v) mixture of a 100 mM N-hydroxysuccinimide (NHS) solution and a 390 mM 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) hydrochloride salt solution in water. Both proteins were diluted to0.05 mg/mL in 20 mM HEPES pH 6.0 for immobilization onto different flow cells of the chip. After a 5 min injection of the protein, remaining reactive groups were deactivated by injecting a 1 M ethanolamine hydrochloride solution in aqueous NaOH (pH 8.5) for 5 min. Different chips were used with immobilization levels ranging between 3,000 and 5,000 response units (RU). To determine kinetic parameters for the binding of compound 6 to human FD and FB, several independent experiments were run. Threefold serial dilutions of compound 6 were prepared ranging from 0.4 to 900 nM. Each solution was injected for 60 s at a flow rate of 30 to 60 μL/min with a dissociation time of at least 1,200 s (parameters identical within one experiment). |
| Affinity data for this assay | |
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