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.
npxskills add adaptyvbio/protein-design-skills--skill binding-characterizationLoading…
| Factor | Choose SPR | Choose BLI |
|---|---|---|
| Sensitivity | Small molecules, fragments (<500 Da) | Large complexes, antibodies |
| Throughput | Low-medium (serial) | High (96-well parallel) |
| Sample purity | Required (clogs fluidics) | Tolerates crude lysates |
| Kinetic resolution | Higher (better for fast kinetics) | Lower |
| Mass transport | More sensitive (may distort kon) | Less sensitive |
| Maintenance | High (fluidics system) | Low (dip-and-read) |
| Sample consumption | Higher (continuous flow) | Lower |
| Cost per experiment | Lower chip cost, higher run cost | Higher tip cost, lower run cost |
| Cause | Mechanism | Solution |
|---|---|---|
| Hydrophobic CDRs | Adsorb to SPR gold/dextran surface | Add 0.05% Tween-20, use CM7 chip with longer dextran |
| Aggregation | Mass transport artifacts in SPR fluidics | Filter sample (0.22μm), reduce ligand density |
| High instability | Degrades during continuous flow | Shorter cycle time, add stabilizers (trehalose 5%) |
| Charge mismatch | Nonspecific binding to charged dextran | Adjust buffer pH ±1 from pI, add BSA 1mg/mL |
| Slow dissociation | Long regeneration needed (damages ligand) | Use BLI (disposable tips) |
| Cause | Mechanism | Solution |
|---|---|---|
| Small analyte | BLI less sensitive for <10 kDa | Use SPR with appropriate chip |
| Weak affinity (KD >10μM) | Fast dissociation in BLI dip | Increase analyte concentration |
| Low expression | Not enough signal | Increase biosensor loading |
Mass transport limitation occurs when analyte cannot diffuse to the surface fast enough to maintain equilibrium. This distorts kinetic parameters.
| Strategy | SPR | BLI |
|---|---|---|
| Reduce ligand density | <200 RU for high-affinity | <0.5 nm shift loading |
| Increase flow rate | 50-100 μL/min | Increase shake speed (1000 rpm) |
| Use oriented immobilization | His-tag capture | Biotinylated ligand |
| Include in fitting | Mass transport model (kt) | Usually less critical |
| Additive | Concentration | Mechanism | Best For |
|---|---|---|---|
| BSA | 0.5-1 mg/mL | Blocks hydrophobic sites | General use |
| Tween-20 | 0.02-0.05% | Prevents surface adsorption | Hydrophobic analytes |
| Trehalose | 1-5% | Stabilizes + blocks | Unstable proteins |
| Sucrose | 5% | BLI-specific blocker | BLI tips |
| Carboxymethyl dextran | 1 mg/mL | Competitive blocking | SPR with charged proteins |
| NaCl | 150-500 mM | Reduces ionic interactions | Charged proteins |
Always include:
| Condition | Targets | Caution |
|---|---|---|
| 10 mM Glycine pH 2.0-2.5 | Most protein-protein | May denature ligand |
| 10 mM Glycine pH 1.5 | Strong interactions | Harsh, limit exposure |
| 1-2 M NaCl | Ionic interactions | Mild, try first |
| 10 mM NaOH | Very stable ligands | Can hydrolyze proteins |
| 10 mM Glycine pH 9-10 | Acid-stable proteins | Can aggregate |
| 10 mM EDTA | His-tag, metal-dependent | Strips Ni-NTA |
| 4 M MgCl2 | Hydrophobic interactions | Check ligand stability |
Symptoms: Two-rate association or dissociation Causes:
Solutions:
Symptoms: Signal increases during dissociation phase Causes:
Solutions:
Symptoms: Signal decreases at high analyte concentrations Causes:
Solutions:
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