Purpose
Determine ka, kdis and KD for the purified binder against biotinylated PD-L1-IgV.
Reagents
- Biotinylated PD-L1-IgV (C-terminal Avi-tag, biotinylated in vitro with BirA)
- Streptavidin (SA) biosensors, hydrated 10 min in kinetics buffer before use
- Kinetics buffer: PBS pH 7.4, 0.02% Tween-20, 0.1% BSA — the BSA cuts nonspecific sensor binding that plain PBS-T lets through
- Regeneration: 10 mM glycine·HCl pH 1.5, 3 × 5 s pulses, then a neutralization dip in kinetics buffer before the next cycle
Procedure
- Hydrate sensors 10 min in kinetics buffer.
- Load PD-L1-IgV to ~0.5-1.0 nm response (target loading rate ≈ 0.5 nm/s — overloading the sensor drives mass-transport-limited kinetics and flattens the apparent koff).
- Baseline 60 s in kinetics buffer.
- Association 120 s against a 2-fold dilution series of binder, 7 points plus a buffer-only reference well (typical top concentration 500-1,000 nM for a first pass, adjusted once a rough KD is in hand).
- Dissociation 300-600 s — extend this if the interaction looks fast (see Caution).
- Regenerate (above) and repeat for the next binder; re-load a fresh sensor if regeneration doesn't return the baseline within ~5% of its starting value.
- Double-reference: subtract both the buffer-only well and a loaded-sensor/no-analyte well before fitting.
- Fit 1:1 Langmuir global (all concentrations, shared ka/kdis) unless the residuals clearly call for a heterogeneous-ligand model.
Caution
De novo minibinders often run fast off-rates. A 60 s dissociation window that looked fine for an antibody will underfill the dissociation curve here, and the fit will overestimate KD (koff pinned near the edge of what the instrument can resolve) — when in doubt, run the dissociation step long and truncate the fit later rather than the other way around.