Binding Characterization: SPR and BLI
SPR vs BLI Decision Matrix
| 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 |
Key differences
SPR (Surface Plasmon Resonance)
- Mechanism: Detects refractive index changes at gold surface
- Surface: Gold chip with dextran matrix (CM5, CM7, etc.)
- Flow: Continuous microfluidics
- Best for: Small molecules, high-affinity, precise kon/koff
BLI (Biolayer Interferometry)
- Mechanism: Measures optical interference pattern shift
- Surface: Fiber optic biosensor tips (SA, Ni-NTA, AHC)
- Flow: Dip-and-read (no microfluidics)
- Best for: High-throughput, crude samples, antibody screening
Troubleshooting: Why BLI works but SPR doesn't
| 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) |
Why SPR works but BLI doesn't
| 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 considerations
Mass transport limitation occurs when analyte cannot diffuse to the surface fast enough to maintain equilibrium. This distorts kinetic parameters.
Symptoms
- Observed kon appears slower than true kon
- Linear association phase (instead of exponential)
- kon varies with ligand density
- Rmax varies with flow rate
When mass transport matters
- High-affinity interactions (kon >10^6 M^-1s^-1)
- High ligand density (>500 RU)
- Slow flow rates (<30 μL/min in SPR)
- Large analytes (slow diffusion)
Mitigation strategies
| 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 |
Nonspecific binding mitigation
Buffer additives (ranked by effectiveness)
| 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 |
pH optimization
- Keep buffer pH at least 1 unit away from analyte pI
- pI near 7: Use pH 6.0 or 8.0 buffer
- Acidic proteins (pI <5): Use neutral or basic buffer
- Basic proteins (pI >9): Use slightly acidic buffer
Reference subtraction
Always include:
- Blank reference channel (no ligand)
- Buffer-only injections
- Non-specific binding controls
Regeneration conditions
SPR regeneration scouting (try in order)
| 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 |
Regeneration protocol
- Start with mildest condition (high salt)
- Test 30s contact time
- Verify complete dissociation (return to baseline)
- Verify retained ligand activity (repeat binding)
- Use shortest effective contact time
BLI tips
- Tips are often disposable (no regeneration needed)
- For reuse: Same conditions as SPR, but shorter exposure
- Anti-His tips: 10 mM Glycine pH 1.5, 30s
- Streptavidin tips: Generally not regenerable
Common artifacts and solutions
Biphasic binding
Symptoms: Two-rate association or dissociation Causes:
- Sample heterogeneity (aggregates)
- Ligand heterogeneity (multiple conformations)
- Avidity effects (bivalent analyte)
Solutions:
- Filter/centrifuge sample
- Use monovalent Fab fragments
- Reduce ligand density
- Fit to heterogeneous model
Negative dissociation
Symptoms: Signal increases during dissociation phase Causes:
- Ligand leaching from surface
- Analyte aggregation on surface
- Reference channel drift
Solutions:
- Use capture antibody instead of direct immobilization
- Increase buffer stringency
- Better reference subtraction
Hook effect
Symptoms: Signal decreases at high analyte concentrations Causes:
- Surface saturation + rebinding suppression
- Crowding effects
Solutions:
- Reduce analyte concentration range
- Reduce ligand density
- Use smaller analyte fragments
Kinetic data quality checklist
Before analysis
- Reference-subtracted properly
- Buffer injection shows flat baseline
- Rmax consistent across concentrations
- No systematic drift during association
- Complete regeneration (return to baseline)
- Duplicate/triplicate injections consistent
Fitting quality
- Residuals randomly distributed (no systematic deviation)
- Chi² < 10% of Rmax (or < 1 RU² for low signals)
- kon and koff errors < 20% of values
- KD from kinetics matches equilibrium KD (within 3-fold)
- Fitted Rmax reasonable (close to theoretical)
Red flags
- kon approaching mass transport limit (>10^7 M^-1s^-1)
- koff faster than data acquisition (< 0.01 s^-1 requires faster sampling)
- Rmax >> theoretical maximum (aggregation or avidity)
- Large difference between kinetic and equilibrium KD
References
Platform comparisons
SPR protocols
Troubleshooting
- 4 Ways to Reduce NSB in SPR - Nicoya
- 3 Ways to Limit Mass Transfer Effects - Nicoya
- Suppressing NSB in BLI - ACS Omega