Thermostability

Thermostability assays measure how proteins respond to increasing temperatures, providing critical developability data for protein therapeutics and industrial applications. These measurements help identify variants with improved stability profiles and predict behavior under storage and processing conditions.

nanoDSF Technology

Our thermostability measurements use nanoDifferential Scanning Fluorimetry (nanoDSF), a highly sensitive technique that monitors protein unfolding through changes in intrinsic tryptophan and tyrosine fluorescence.

1

Sample Preparation

Purified proteins are prepared in standard buffer conditions or customer-specified formulations.

2

Temperature Ramping

Samples are subjected to controlled temperature increases while monitoring fluorescence changes.

3

Melting Point Determination

The midpoint of the unfolding transition (Tm) is determined from the fluorescence data.

Key Measurements

Primary Stability Metrics

Units: °C Meaning: Temperature at which 50% of proteins are unfolded

The most commonly reported stability metric, representing the temperature at the midpoint of the unfolding transition.

Typical ranges:

  • High stability: >70°C
  • Good stability: 50-70°C
  • Moderate stability: 40-50°C
  • Poor stability: <40°C

Advanced Analysis

Unfolding Kinetics

  • Rate of unfolding at different temperatures
  • Activation energy calculations
  • Predictive modeling of stability

Multi-Domain Analysis

  • Multiple transition identification
  • Domain-specific stability assessment
  • Cooperative vs. independent unfolding

Applications

Developability Assessment

Evaluate protein stability for pharmaceutical development, identifying variants suitable for therapeutic applications.

Formulation Optimization

Test protein stability under different buffer conditions, pH levels, and additive concentrations.

Stability Engineering

Guide protein engineering efforts to improve thermal stability through rational or evolutionary approaches.

Quality Control

Monitor protein stability during development, manufacturing, and storage processes.

Experimental Conditions

Standard Conditions

Buffer Systems

  • PBS (pH 7.4) - Standard physiological conditions
  • Histidine buffer (pH 6.0) - Common formulation buffer
  • Custom buffers - Customer-specified conditions

Temperature Range

  • Typical: 20°C to 95°C
  • Customizable based on protein characteristics
  • High-resolution scanning (0.3°C/min)

Custom Formulations

pH Studies

  • Multiple pH conditions (4.0-8.0)
  • Buffer system optimization
  • pH stability profiling

Additive Screening

  • Stabilizing agents (trehalose, sucrose, arginine)
  • Destabilizing conditions (urea, guanidine)
  • Excipient compatibility testing

Data Analysis

Curve Fitting

Transition Modeling

  • Two-state unfolding models
  • Multi-transition analysis for complex proteins
  • Thermodynamic parameter extraction

Statistical Analysis

  • Replicate analysis and error estimation
  • Comparative statistics between variants
  • Quality control metrics

Comparative Analysis

Ranking and Selection

  • Tm-based variant ranking
  • Multi-parameter optimization
  • Structure-stability relationships

Predictive Modeling

  • Stability under storage conditions
  • Accelerated stability predictions
  • Formulation stability forecasting

Integration with Other Assays

Thermostability measurements complement other characterization assays:

Binding-Stability Relationships

  • Correlation between binding affinity and thermal stability
  • Trade-off analysis for optimization decisions
  • Stability-function balance assessment

Developability Profiles

  • Combined analysis with aggregation propensity
  • Viscosity and stability correlations
  • Comprehensive developability scoring

When to Use Thermostability Assays

Quality Control

Measurement Validation

Technical Replicates

  • Multiple measurements per sample
  • Statistical analysis of reproducibility
  • Quality metrics for each measurement

Reference Standards

  • Known stability controls
  • Instrument performance verification
  • Batch-to-batch consistency monitoring

Data Quality Assessment

Signal Quality

  • Fluorescence signal intensity validation
  • Baseline stability assessment
  • Transition clarity evaluation

Fit Quality

  • Goodness of fit statistics
  • Residual analysis
  • Parameter confidence intervals

Results Package

Your thermostability results include:

Quantitative Data

  • Melting temperatures (Tm) with confidence intervals
  • Onset temperatures and transition widths
  • Comparative ranking of all variants tested

Visual Analysis

  • Melting curves showing fluorescence vs. temperature
  • Derivative plots highlighting transition points
  • Comparative overlays for easy variant comparison

Analysis Reports

  • Stability assessment with recommendations
  • Structure-stability correlations when structural data available
  • Formulation recommendations based on stability profiles

Getting Started

Ready to assess protein thermostability?

  1. Select your protein variants for stability assessment
  2. Choose experimental conditions (standard or custom buffers)
  3. Configure your experiment through the Foundry Portal
  4. Review conditions with our technical team

Thermostability assays are particularly valuable when combined with binding measurements. Consider running both assays to understand binding-stability trade-offs in your optimization campaigns.