Biacore t200 surface plasmon resonance system

The affinity and kinetics of interactions between whole-cell N. gonorrhoeae and glycans were investigated using the Biacore T200 surface plasmon resonance system (General Electric). Bacteria were grown as described above, harvested into PBS, and washed twice (2,000 × g, 5 min, room temperature), after which bacteria were fixed in 2.5% (vol/vol) formaldehyde for 10 min at room temperature. Bacterial cells were pelleted at 2,000 × g and washed three times in PBS to remove residual fixative. Fixed bacteria were made up to an OD₆₀₀ of 0.1 (∼1 × 10⁷ CFU/ml) in 10 mM sodium acetate buffer, pH 4 (optimal pH was determined by a series of pH scout experiments), and immobilized on flow cells 2, 3, and 4 at a flow rate of 5 μl/min for total contact time of 1,200 s. Flow cell 1 (no-bacteria control) was used as a reference for nonspecific interactions between glycans and the surface of the chip. Single-cycle kinetics were used to generate the K_D of interactions between whole-cell N. gonorrhoeae and free glycans. Analytes were prepared and run in degassed PBS⁺ at a flow rate of 30 μl/min for total contact times of 90 s and 600 s of dissociation. Each analyte was run twice per flow cell. All data analysis was performed using Biacore T200 Evaluation software (v3.0; GE).

Affinity assays of Evasin-3 variants were performed using BIAcore T200 surface plasmon resonance system (GE Healthcare). All assays were carried out in PBS buffer, pH 7.4, supplemented with 0.05% Tween 20 as running buffer. 70 response units of chemically synthetized CXCL8-Biotin was immobilized on a SAHC 200M sensorchip (Xantec) according to the manufacturer's protocol. Concentration series of Evasin-3 variants, ranging from 1 μm to 2 nm, were prepared by 2-fold dilution of 50 μm stock solutions. Association and dissociation kinetics were monitored at flow rate 30 μl/min for 250 and 300 s, respectively. The data were analyzed using BIAcore T200 software and BIAEvaluation software (GE Healthcare) using a steady-state affinity equation with a linear component.