Biophysical Characterization of
Protein:Ligand Interactions

Biophysical methods such as TSA, SPR, or ITC are orthogonal to commonly used high-throughput screening methods and can provide confirmation of the molecular interaction of HTS hits. Each method provides different data to guide discovery and development.

Thermal Stability Analysis (TSA) OR DIFFERENTIAL SCANNING FLUORIMETRY (DSF)

  • Measures the temperature at which a protein unfolds (Tm)
  • Measures ligand-induced changes to thermal stability
  • Ranks ligands by affinity; is powerful method for fragment-based screening
  • Maps protein stability in various buffer conditions
  • Has minimal protein requirements
  • Can screen 10,000 compound per week

Surface Plasmon Resonance (SPR)

  • Measures association and dissociation rates (Ka and Kd); enables calculation of the equilibrium dissociation constant (KD)
  • Elucidates the interaction kinetics between a protein and its ligand
  • Requires attachment of the protein or the ligand to a biosensor chip

Isothermal Titration Calorimetry (ITC)

  • Provides a complete thermodynamic profile of protein:ligand interactions
  • Protein and ligand remain  free of modification, tags, or coupling
  • Quantitatively determines equilibrium binding affinity (Ka), binding stoichiometry, and enthalpy of binding (ΔH)
  • Can determine entropy (ΔS), free energy of binding (ΔG), and dissociation constant (Kd) by extension