Thermal Shift Analysis

The thermal shift analysis (TSA) is a thermal denaturation assay for elucidating conditions that stabilize a protein in solution. Protein destabilization and melting is measured as an increase in fluorescence as hydrophobic binding sites within the protein become exposed during unfolding and are able to bind a fluorescent dye in the assay mixture. The melting temperature of a protein (Tm) is identified as the temperature at which 50% of the protein is unfolded.  The assay is performed in plate format, allowing a large number of conditions (buffers, ligands, etc.) to be screened simultaneously. Conditions that cause the Tmto shift to a higher temperature are identified as stabilizing.  

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Available thermal shift assays:

  • Tm determination for batch to batch validation
  • Buffer screening for formulation optimization
  • Ligand binding studies
  • Protein:protein binding studies
  • Construct stability comparison
  • Thermal Shift Assay Melt Curve
  • Thermal Shift Assay Melt Peak

Results of a thermal shift assay for a representative protein formulated in a series of buffers that range in pH from 5-9.5.  The left panel represents the raw fluorescence data and the right panel shows to the first derivative curves.  The peak minimum of the first derivative curves corresponds to the protein melting temperature.  For the example shown, the stability of the protein increases with decreasing pH.