SPR (Surface Plasmon Resonance)
Real-time, label-free binding kinetics and affinity measurements using Surface Plasmon Resonance technology
Surface Plasmon Resonance (SPR) is a powerful, label-free optical technique for measuring biomolecular interactions in real-time. Our SPR platform provides precise kinetic and thermodynamic characterization of protein-protein, protein-small molecule, and protein-nucleic acid interactions.
Technology Principle
SPR detects changes in refractive index near a sensor surface when molecules bind to immobilized ligands. This label-free detection enables real-time monitoring of association and dissociation events with high sensitivity and precision.
Surface Plasmons
Electromagnetic waves at metal-dielectric interface
- Gold sensor chip surface
- Angle-dependent resonance
- Refractive index sensitivity
- Real-time detection
Binding Detection
Mass changes during molecular interactions
- Association kinetics (kon)
- Dissociation kinetics (koff)
- Equilibrium binding (KD)
- Thermodynamic parameters
Key Measurements
Real-time binding kinetics with comprehensive rate constant determination
Parameters measured:
- Association rate constant (kon): 10³ to 10⁹ M⁻¹s⁻¹
- Dissociation rate constant (koff): 10⁻⁶ to 10⁻¹ s⁻¹
- Equilibrium dissociation constant (KD = koff/kon)
- Residence time (RT = 1/koff)
Applications:
- Drug discovery and development
- Antibody characterization
- Enzyme-substrate interactions
- Protein-protein interactions
SPR provides the most comprehensive kinetic characterization available for biomolecular interactions.
Real-time binding kinetics with comprehensive rate constant determination
Parameters measured:
- Association rate constant (kon): 10³ to 10⁹ M⁻¹s⁻¹
- Dissociation rate constant (koff): 10⁻⁶ to 10⁻¹ s⁻¹
- Equilibrium dissociation constant (KD = koff/kon)
- Residence time (RT = 1/koff)
Applications:
- Drug discovery and development
- Antibody characterization
- Enzyme-substrate interactions
- Protein-protein interactions
SPR provides the most comprehensive kinetic characterization available for biomolecular interactions.
Equilibrium binding analysis for steady-state measurements
Measurement range:
- KD values: pM to mM range
- Concentration series analysis
- Saturation binding curves
- Competitive binding assays
Advantages:
- Model-independent KD determination
- Low sample consumption
- Multiple analyte screening
- Concentration-response curves
Equilibrium analysis is particularly useful for weak binders or when kinetic analysis is challenging.
Selectivity and cross-reactivity assessment
Applications:
- Antibody specificity profiling
- Drug selectivity screening
- Off-target interaction detection
- Biosensor development
Methods:
- Multi-cycle kinetics
- Single-cycle kinetics
- Competition assays
- Inhibition studies
Specificity studies are essential for therapeutic development and diagnostic applications.
Experimental Design
Surface Chemistry Selection
Choose appropriate immobilization strategy based on ligand properties and experimental goals.
Amine Coupling
Amine Coupling
EDC/NHS chemistry for proteins with accessible primary amines
Advantages:
- Stable covalent attachment
- High ligand density possible
- Well-established protocols
- Broad applicability
Considerations:
- May affect protein orientation
- Multiple attachment points
- Requires primary amines
- pH sensitivity during coupling
Thiol Coupling
Thiol Coupling
Maleimide or disulfide chemistry for cysteine-containing proteins
Advantages:
- Site-specific attachment
- Controlled orientation
- Mild coupling conditions
- Reduced heterogeneity
Applications:
- Antibody Fab fragments
- Engineered cysteine proteins
- Peptide immobilization
- Oriented immobilization
Capture Methods
Capture Methods
His-tag, biotin-streptavidin, or antibody capture
Advantages:
- Reversible immobilization
- Native protein orientation
- Easy regeneration
- Minimal protein modification
Applications:
- Tagged protein analysis
- Protein complex studies
- Fragile protein interactions
- Screening applications
Ligand Immobilization
Optimize ligand density and activity for reliable kinetic measurements.
Key parameters:
- Ligand density: 100-2000 RU (response units)
- Activity assessment: analyte binding capacity
- Orientation verification: functional binding
- Stability testing: long-term performance
High ligand densities can lead to mass transport limitations and artifacts in kinetic analysis.
Analyte Preparation
Prepare analyte samples with appropriate concentration ranges and buffer conditions.
Concentration series:
- Typical range: 0.1x to 10x KD
- 5-8 concentration points
- 3-fold dilution series
- Buffer matching critical
Buffer considerations:
- HEPES, PBS, or Tris-based buffers
- Physiological salt concentrations
- pH 7.0-7.5 (typically)
- Additive compatibility assessment
Buffer composition should match immobilization conditions to minimize bulk refractive index changes.
Measurement Protocol
Execute binding experiments with appropriate controls and reference subtraction.
Typical protocol:
- Baseline stabilization (2-5 minutes)
- Analyte injection (association phase)
- Buffer injection (dissociation phase)
- Regeneration (if applicable)
- Reference subtraction
Include reference flow cells and negative controls to ensure data quality and specificity.
Data Analysis and Modeling
Kinetic Modeling
1:1 Langmuir Model
1:1 Langmuir Model
Simple bimolecular interaction (A + B ⇌ AB)
Assumptions:
- Single binding site
- Homogeneous analyte
- No mass transport limitation
- Reversible binding
Applications:
- Simple protein-protein interactions
- Small molecule binding
- Antibody-antigen interactions
- Initial screening studies
Equation: dR/dt = kon × Ca × (Rmax - R) - koff × R
Two-State Model
Two-State Model
Conformational change upon binding (A + B ⇌ AB ⇌ AB*)
Features:
- Initial binding step
- Conformational rearrangement
- Improved fit quality
- Biological relevance
Applications:
- Enzyme-substrate interactions
- Allosteric binding
- Conformational changes
- Complex binding mechanisms
Heterogeneous Ligand Model
Heterogeneous Ligand Model
Multiple binding sites or populations
Features:
- Multiple KD values
- Different binding capacities
- Population analysis
- Improved data fitting
Applications:
- Heterogeneous ligand preparations
- Multiple binding sites
- Avidity effects
- Complex interactions
Quality Control Metrics
Kinetic Quality
Kinetic parameter reliability
- Chi-squared (χ²) values <10
- Residuals analysis
- Parameter standard errors
- Model comparison statistics
Experimental Quality
Data collection quality
- Signal-to-noise ratio >20
- Baseline stability (±2 RU)
- Injection reproducibility
- Reference subtraction quality
Binding Quality
Interaction specificity
- Specific vs. non-specific binding
- Dose-response relationships
- Saturation behavior
- Competition studies
Surface Quality
Sensor surface performance
- Ligand activity maintenance
- Regeneration efficiency
- Surface stability
- Drift assessment
Advanced SPR Techniques
Multi-Cycle vs. Single-Cycle Kinetics
Individual injections for each analyte concentration
Advantages:
- Complete dissociation between cycles
- Full kinetic information per concentration
- Better curve fitting
- Traditional approach
Applications:
- High-quality kinetic analysis
- Method development
- Detailed characterization
- Publication-quality data
Considerations:
- Longer analysis time
- More sample consumption
- Surface regeneration required
- Potential surface degradation
Individual injections for each analyte concentration
Advantages:
- Complete dissociation between cycles
- Full kinetic information per concentration
- Better curve fitting
- Traditional approach
Applications:
- High-quality kinetic analysis
- Method development
- Detailed characterization
- Publication-quality data
Considerations:
- Longer analysis time
- More sample consumption
- Surface regeneration required
- Potential surface degradation
Sequential injections without regeneration
Advantages:
- Faster analysis
- Reduced sample consumption
- No regeneration artifacts
- Better for fragile surfaces
Applications:
- High-throughput screening
- Fragile interaction analysis
- Limited sample availability
- Routine analysis
Limitations:
- Less kinetic information
- Requires stable surfaces
- Model-dependent analysis
- Limited concentration range
Specialized Applications
Fragment Screening
Fragment Screening
Low molecular weight compound screening for drug discovery:
Challenges:
- Low molecular weight signals
- Weak binding affinities
- High compound concentrations
- Solubility limitations
Solutions:
- High ligand density surfaces
- Optimized buffer systems
- Statistical analysis methods
- Hit validation protocols
Throughput: 100-1000 compounds per day
Antibody Characterization
Antibody Characterization
Comprehensive antibody analysis including epitope binning:
Measurements:
- Binding kinetics and affinity
- Epitope mapping studies
- Species cross-reactivity
- Isotype comparisons
Applications:
- Therapeutic antibody development
- Diagnostic antibody selection
- Biosimilar characterization
- Quality control testing
Protein-Protein Interactions
Protein-Protein Interactions
Complex formation and interaction network analysis:
Capabilities:
- Binary interaction analysis
- Competitive binding studies
- Ternary complex formation
- Allosteric effect detection
Applications:
- Signaling pathway studies
- Drug mechanism of action
- Protein function analysis
- Interaction validation
Integration with Other Technologies
SPR measurements complement other biophysical techniques:
Structural Studies
Validate binding sites identified by crystallography or NMR
- Confirm solution-phase binding
- Quantify binding strength
- Assess binding kinetics
- Structure-activity relationships
Cell-Based Assays
Correlate molecular binding with cellular activity
- Binding vs. functional potency
- Selectivity confirmation
- Mechanism validation
- Dose-response relationships
Computational Modeling
Validate predictions from molecular modeling
- Docking score correlation
- Binding mode validation
- Affinity predictions
- Drug design optimization
Other Biophysics
Orthogonal binding measurements
- ITC thermodynamics
- BLI kinetics comparison
- MST binding confirmation
- NMR interaction mapping
Quality Assurance and Compliance
Method Validation
Precision and Accuracy
Assess method repeatability and reproducibility across operators and instruments.
Acceptance criteria:
- Kinetic rate constants: CV <20%
- Equilibrium KD values: CV <30%
- Inter-analyst precision: CV <25%
- Long-term reproducibility: CV <35%
Range and Linearity
Establish working ranges for kinetic and equilibrium measurements.
Kinetic range:
- kon: 10³ to 10⁸ M⁻¹s⁻¹
- koff: 10⁻⁵ to 10⁻¹ s⁻¹
- KD: pM to μM range
Linearity assessment:
- Concentration vs. response
- Rate vs. concentration plots
- Binding capacity analysis
Robustness Testing
Evaluate method performance under varied conditions.
Test parameters:
- Buffer composition variations
- Temperature fluctuations (±2°C)
- Flow rate changes (±10%)
- Injection volume variations (±5%)
Regulatory Compliance
ICH guidelines for bioanalytical method validation:
- Accuracy and precision requirements
- Selectivity and specificity
- Stability and robustness
- Quality control procedures
Data integrity requirements:
- 21 CFR Part 11 compliance
- Audit trail maintenance
- Electronic signature controls
- Data backup and archival
SPR measurements require careful attention to mass transport effects, which can lead to apparent kinetic artifacts if not properly controlled.
Troubleshooting Common Issues
Mass Transport Limitations
Mass Transport Limitations
Symptoms: Concentration-dependent kinetics, curved Scatchard plots, reduced apparent kon
Solutions:
- Reduce ligand density (target 100-500 RU)
- Increase flow rate (≥30 μL/min)
- Use shorter contact time
- Consider single-cycle kinetics
Prevention:
- Optimize ligand immobilization levels
- Use appropriate flow rates from the start
- Monitor for concentration-dependent artifacts
Bulk Refractive Index Effects
Bulk Refractive Index Effects
Symptoms: Large baseline shifts, poor reference subtraction, drift
Solutions:
- Match analyte and running buffer exactly
- Include buffer blanks in sequence
- Use appropriate reference flow cells
- Filter all solutions (0.22 μm)
Prevention:
- Careful buffer preparation and matching
- Consistent sample preparation procedures
- Regular instrument maintenance
Non-Specific Binding
Non-Specific Binding
Symptoms: High responses to negative controls, poor specificity
Solutions:
- Optimize surface chemistry and blocking
- Include appropriate negative controls
- Use competitor molecules
- Increase salt concentration moderately
Prevention:
- Proper surface preparation and blocking
- Include specificity controls in all experiments
- Use well-characterized negative control proteins
Poor Regeneration
Poor Regeneration
Symptoms: Decreased binding capacity over cycles, baseline drift
Solutions:
- Optimize regeneration conditions
- Use milder regeneration (shorter time, lower concentration)
- Consider alternative regeneration agents
- Replace sensor chip if necessary
Prevention:
- Test regeneration conditions during method development
- Monitor binding capacity over time
- Use appropriate regeneration protocols
Best Practices and Recommendations
Experimental Design
- Control experiments: Always include negative controls and reference surfaces
- Concentration series: Use appropriate concentration ranges (0.1x to 10x KD)
- Buffer optimization: Match all buffer conditions between ligand and analyte
- Surface validation: Confirm ligand activity and orientation
Data Quality
- Baseline stability: Ensure stable baselines before and after injections
- Reference subtraction: Use appropriate reference flow cells
- Reproducibility: Include replicate measurements for key interactions
- Model validation: Compare different kinetic models and assess fit quality
Method Development
- Ligand optimization: Test different immobilization strategies and densities
- Kinetic validation: Confirm mass transport-free conditions
- Specificity testing: Include comprehensive specificity panels
- Robustness assessment: Test method performance under varied conditions
Regular instrument maintenance and calibration are essential for consistent, high-quality SPR results. Our team provides comprehensive training and support for SPR method development and validation.