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Assay Method Information |
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| | SPR Assay |
| Description: | Recombinant ezrin (Hypermol) was diluted to 70 mg/mL in sodium acetate buffer (pH 5.5, 10 mM) immobilized for 350 s on a CM5 Sensor Chip (Biacore) by standard amine coupling to yield around 10,000 response units. The reference flow cell was prepared exactly as the ligand flow cell, although sodium acetate buffer only (pH 5.5, 10 mM) was used instead of recombinant ezrin. Binding interactions were monitored at 25 °C with a flow rate of 30 mL/min in HBSEP/1% DMSO as running buffer. Disorazole A was injected at 0, 10, 20, 40, 60, and 80 mM. Disorazole Z was injected at 0, 20, 40, 60, 80, and 100 mM. The contact time for each compound was 120 s and the dissociation time 500 s. To rule out non-specific binding, a negative control protein was immobilized on a CM5 Sensor Chip (Biacore) by standard amine coupling: BSA (Sigma) was diluted to 70 mg/mL in sodium acetate buffer (pH 4.5, 10 mM) and immobilized for 90 s to yield around 5,000 response units. The reference flow cell was prepared with sodium acetate buffer (pH 4.5). |
| Affinity data for this assay | |
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