| Assay Method Information | |
| | Target Residence Time of Factor B Inhibitors Determined by Surface Plasmon Resonance (SPR) |
| Description: | Table 2: Biacore 8 k instrument was primed using 1× PBS-P+ buffer before docking a Cytiva NTA chip. Recombinant human Factor B catalytic domain were immobilized on a NTA chip to a level of about 5000 resonance units (RU) using 1× PBS-P+ buffer [20 mM phosphate buffer with 2.7 mM KCl, 137 mM NaCl, and 0.05% (v/v) Tween-20]. The protein ligand was further crosslinked to sensorchip surface by amine coupling kit. Immobilization and binding experiment were performed at room temperature.After changing buffer to 1× PBS-P+ buffer with 2% (v/v) DMSO, a pre-run was performed for a period of at least 30 min at a flow rate of 30 μl/min to obtain a stable surface. The kinetic constants of the compounds were determined by single-cycle kinetics with six consecutive injections (or multi-cycle kinetics with eight consecutive injections) with an increasing compound concentration in ranges of 0.8-200 nM, 12.5-400 nM, 4.1-1,000 nM or 41-10,000 nM depending on the potency. Single-cycle kinetics experiments were performed with an association time of 60 s per concentration and a dissociation time of 300 s (or a dissociation time of 120 s for multi-cycle kinetics experiments). A flow rate of 30 μl/min was used. A blank run with the same conditions was performed before the compound was injected. |
| Affinity data for this assay | |
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