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.

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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

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Key Measurements

Real-time binding kinetics with comprehensive rate constant determinationParameters 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.

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Experimental Design

1

Surface Chemistry Selection

Choose appropriate immobilization strategy based on ligand properties and experimental goals.
2

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.
3

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.
4

Measurement Protocol

Execute binding experiments with appropriate controls and reference subtraction.Typical protocol:
  1. Baseline stabilization (2-5 minutes)
  2. Analyte injection (association phase)
  3. Buffer injection (dissociation phase)
  4. Regeneration (if applicable)
  5. Reference subtraction
Include reference flow cells and negative controls to ensure data quality and specificity.

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Data Analysis and Modeling

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Kinetic Modeling

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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

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Advanced SPR Techniques

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Multi-Cycle vs. Single-Cycle Kinetics

Individual injections for each analyte concentrationAdvantages:
  • 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

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Specialized Applications

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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

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Quality Assurance and Compliance

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Method Validation

1

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%
2

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
3

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%)

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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.

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Troubleshooting Common Issues

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Best Practices and Recommendations

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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

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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

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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.