Study the thermal stability of proteins and investigate buffer conditions, ligands, cofactors and drugs affecting this stability to rapidly identify promising protein formulation and complexes for further structural characterization
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Thermal shift assay is a thermodenaturation assay to monitor the thermal stability of proteins and investigate factors affecting this stability. This rapid and simple technique is used in high-throughputmode to screen optimalbuffer conditions, ligands, cofactors and drugs for purified proteins. Two methods to monitor protein denaturation are available: a differential scanning fluorimetry (DSF) method and a differential static light scattering method (DSLS). The optimization of proteins solubility and stability properties improvesthe success rate of their structural studies. Changes in the thermal stability of the protein–ligand or protein-peptide complexes relative to the stability of the protein alone allow to rapidly identify promising complexes for further structural characterization and to assign functions.
Thermal shift assay is a microplate binding assay to quickly screen buffer/ligand conditions by monitoring thermal melting curves. The differential scanning fluorimetry (DSF) method uses an environmentally sensitive dye. The fluorescence signal increases as a function of temperature in the hydrophobic environment introduced by protein unfolding during heat-denaturation. The plot of the fluorescence signal as a function of temperature is a sigmoid curve. The inflection point is usually referred to as the melting temperature (Tm) where 50% of the protein is unfolded. An increase in Tm values will identify stabilizing conditions. Hydrophobic proteins cannot be analyzed using DSF due to high fluorescence backgrounds. With the differential static light scattering, aggregation of unfolded proteins upon heat-denaturation is monitored by light scattering. In this latter case, the inflection point of the sigmoid curve is called Tagg. Very small amounts of protein sample (1-15 µg per well) areneeded to set up a thermal shift assay.