Surface plasmon resonance (SPR) affinity assay

For the SPR affinity assay, a 1× HBS-EP⁺ buffer is formulated as the running buffer for the experiment. The hNogo-B-N protein is diluted to a concentration of 5 μg/mL using the running buffer and immobilized on the Fc2 channel of the SPR instrument via ligand capture for a duration of 40 s, until a signal of approximately 290 resonance units (RU) is achieved. The analyte, proGCG protein, is diluted to a starting concentration of 4000 nM in the running buffer and subjected to a continuous two-fold dilution series. The analyte is then injected over both the experimental and reference channels, with an association phase lasting 90 s at a flow rate of 30 μL/min, followed by a dissociation phase of 120 s. The system is regenerated using a 10 mM glycine-HCl (pH 1.5) regeneration buffer, applied for 30 s at the same flow rate to remove bound analytes and restore the sensor surface activity. Data analysis involves the kinetic evaluation of at least five consecutive concentration points, employing either a 1:1 binding model or a steady-state analysis approach to determine the affinity constants between the ligand and the analyte.