By name: binding-characterization description: > Guidance for SPR and BLI binding characterization experiments. Use when: (1) Planning binding kinetics experiments, (2) Troubleshooting poor/no binding signal, (3) Interpreting kinetic data artifacts, (4) Choosing between SPR vs BLI platforms. license: MIT category: experimental tags: [binding, spr, bli, validation] source: https://github.com/adaptyvbio/protein-design-skills
Binding Characterization: SPR and BLI
SPR vs BLI Decision Matrix
| Factor | Choose SPR | Choose BLI |
|---|---|---|
| Sensitivity | Small molecules (<500 Da) | Large complexes, antibodies |
| Throughput | Low-medium (serial) | High (96-well parallel) |
| Sample purity | Required (clogs fluidics) | Tolerates crude lysates |
| Kinetic resolution | Higher (fast kinetics) | Lower |
| Mass transport | More sensitive | Less sensitive |
| Maintenance | High (fluidics) | Low (dip-and-read) |
Troubleshooting Matrix
| Problem | Mechanism | Solution |
|---|---|---|
| Hydrophobic CDRs adsorb to SPR surface | Gold/dextran surface | Add 0.05% Tween-20, use CM7 chip |
| Aggregation in SPR fluidics | Mass transport artifacts | Filter sample (0.22μm), reduce ligand density |
| Protein degrades during continuous flow | High instability | Shorter cycle time, add trehalose 5% |
| Small analyte (<10 kDa) on BLI | Low signal | Use SPR instead |
| Weak affinity (KD >10μM) on BLI | Fast dissociation | Increase analyte concentration |
Mass Transport Considerations
Symptoms of mass transport limitation
- Linear association phase (not exponential)
- kon varies with ligand density
- Rmax varies with flow rate
Mitigation
- SPR: Reduce ligand density (<200 RU), increase flow rate (50-100 μL/min)
- BLI: Reduce loading (<0.5 nm shift), increase shake speed (1000 rpm)
Regeneration Conditions (SPR)
| Condition | Targets | Caution |
|---|---|---|
| 10 mM Glycine pH 2.0-2.5 | Most protein-protein | May denature ligand |
| 1-2 M NaCl | Ionic interactions | Mild, try first |
| 10 mM NaOH | Very stable ligands | Can hydrolyze proteins |
| 10 mM EDTA | His-tag, metal-dependent | Strips Ni-NTA |
Kinetic Data Quality Checklist
- Reference-subtracted properly
- Buffer injection shows flat baseline
- Chi² < 10% of Rmax
- kon and koff errors < 20%
- KD from kinetics matches equilibrium KD (within 3-fold)
Red Flags
- kon approaching mass transport limit (>10⁷ M⁻¹s⁻¹)
- Rmax >> theoretical maximum (aggregation or avidity)
- Large difference between kinetic and equilibrium KD