Thermal Shift Assay (TSA)

Thermal Shift Assay (TSA) is a versatile technique for assessing protein stability under different conditions. Using extrinsic fluorescent dyes, TSA monitors thermal unfolding by detecting fluorescence changes as proteins denature. The assay supports protein–ligand interaction studies, buffer optimization, formulation development, identification of stabilizing conditions, and broader drug-discovery workflows.

Proteins normally exist in a native, folded state. As temperature increases, molecular motion intensifies and proteins begin to unfold, exposing hydrophobic regions. An extrinsic fluorescent dye binds to these newly exposed regions, causing fluorescence intensity to increase. When the temperature reaches the melting temperature (T_m), unfolded proteins aggregate and fewer hydrophobic sites remain available for dye binding. The dye returns to the aqueous environment and fluorescence is quenched, producing a characteristic increase–decrease melting curve. Plotting fluorescence intensity against temperature enables determination of the protein T_m.

Principles of the Thermal Shift Assay. Fluorescence changes caused by dye binding and quenching are used to generate a protein melting curve and determine T_m.