nanoDSF
Label-free protein stability analysis using nanoDifferential Scanning Fluorimetry for thermal stability characterization
nanoDSF (nanoDifferential Scanning Fluorimetry) is a cutting-edge, label-free technique for analyzing protein thermal stability. Our platform uses nanoDSF to provide precise measurements of protein melting temperatures and aggregation propensities.
Technology Overview
nanoDSF monitors the intrinsic fluorescence of aromatic amino acids (tryptophan and tyrosine) as proteins unfold during controlled temperature ramping. This label-free approach provides detailed insights into protein stability without the need for external dyes or modifications.
Intrinsic Fluorescence
Tryptophan and tyrosine fluorescence changes upon unfolding
- Wavelength shifts indicate structural changes
- Intensity changes reflect environment changes
- Native protein monitoring without labels
Light Scattering
Static light scattering detects aggregation
- Real-time aggregation monitoring
- Aggregation onset temperature (Tagg)
- Aggregation kinetics analysis
Key Measurements
Melting Temperature (Tm)
The temperature at which 50% of proteins are unfolded, determined from fluorescence ratio changes.
Applications:
- Protein stability comparison
- Buffer optimization
- Mutation impact assessment
- Formulation development
Our nanoDSF system can detect multiple Tm values for proteins with multiple domains.
Aggregation Temperature (Tagg)
The temperature at which protein aggregation begins, measured by light scattering increase.
Applications:
- Aggregation propensity assessment
- Storage condition optimization
- Formulation stability testing
- Quality control metrics
The difference between Tm and Tagg indicates the stability window where proteins are unfolded but not aggregated.
Unfolding Kinetics
Real-time monitoring of unfolding processes and intermediates.
Applications:
- Unfolding mechanism studies
- Intermediate state identification
- Kinetic parameter determination
- Pathway analysis
Experimental Capabilities
Temperature Range and Control
- Range: 15°C to 95°C
- Ramp rate: 0.1°C to 10°C per minute
- Precision: ±0.1°C temperature accuracy
- Hold steps: Isothermal holds for kinetic studies
Sample Requirements
Volume: 10 μL per sample Concentration: 0.1-2.0 mg/mL Buffer: Any aqueous buffer system Throughput: 48 samples per run
Typical conditions:
- pH 6.0-8.5
- Salt concentration: 50-500 mM
- Compatible with most buffer systems
Volume: 10 μL per sample Concentration: 0.1-2.0 mg/mL Buffer: Any aqueous buffer system Throughput: 48 samples per run
Typical conditions:
- pH 6.0-8.5
- Salt concentration: 50-500 mM
- Compatible with most buffer systems
Volume: 2-5 μL per sample Concentration: 0.05-0.5 mg/mL Enhanced detection: For low-fluorescence proteins Special applications: Precious samples, limited material
Features:
- Extended measurement time
- Signal averaging
- Background correction
- Noise reduction algorithms
Parallel analysis: Multiple buffer conditions Automated comparisons: Side-by-side stability assessment Optimization: Rapid condition screening Statistical analysis: Automated data comparison
Applications:
- Formulation development
- Storage optimization
- pH stability mapping
- Salt effect analysis
Data Analysis and Interpretation
Fluorescence Analysis
Light Scattering Analysis
Aggregation Detection:
- Onset temperature determination
- Aggregation rate analysis
- Particle size distribution
- Reversibility assessment
Quality Metrics:
- Sample homogeneity
- Pre-existing aggregation
- Measurement reliability
- Data quality assessment
Applications
Protein Engineering
Stability optimization through rational design
- Mutation impact assessment
- Stabilizing mutation identification
- Design-build-test cycles
- Structure-stability relationships
Formulation Development
Buffer and storage optimization
- pH stability profiles
- Salt effect analysis
- Excipient screening
- Storage condition optimization
Quality Control
Batch consistency monitoring
- Stability acceptance criteria
- Process control metrics
- Lot release testing
- Degradation monitoring
Drug Development
Biopharmaceutical stability assessment
- Developability assessment
- Forced degradation studies
- Stress testing protocols
- Shelf-life determination
Advanced Analysis Features
Comparative Studies
- Multi-sample analysis: Up to 48 samples per experiment
- Statistical comparison: Automated significance testing
- Trend analysis: Systematic variation assessment
- Ranking systems: Stability-based prioritization
Kinetic Analysis
- Unfolding kinetics: Rate constant determination
- Aggregation kinetics: Time-dependent analysis
- Temperature-dependent rates: Arrhenius analysis
- Mechanism elucidation: Pathway determination
Data Integration
- Database storage: Systematic data management
- Trend tracking: Historical comparison
- Correlation analysis: Structure-stability relationships
- Predictive modeling: Stability prediction algorithms
Integration with Other Technologies
nanoDSF data complements other analytical methods:
Thermal shift assays combined with binding measurements
- Ligand-induced stabilization
- Binding affinity estimation
- Allosteric effect detection
- Drug target validation
Thermal shift assays combined with binding measurements
- Ligand-induced stabilization
- Binding affinity estimation
- Allosteric effect detection
- Drug target validation
Correlation with structural data
- HPLC-SEC stability correlation
- Aggregation mechanism insights
- Structural integrity assessment
- Folding pathway analysis
Activity-stability relationships
- Thermostability vs. activity
- Optimal temperature determination
- Storage impact on function
- Stability-function trade-offs
Quality Control and Validation
Proteins lacking aromatic amino acids may show limited fluorescence changes. Alternative approaches or protein engineering may be required for such cases.
nanoDSF works best for proteins with at least one tryptophan or several tyrosine residues. Our team can assess protein suitability and suggest optimization strategies.