Label-Free Target Identification and Confirmation Using Thermal Stability Shift Assays

Target identification presents one of the biggest challenges to chemical genomic approaches. In recent years, several methods have been applied for target identification and validation in plant cells. Here, we describe a label-free method based on the thermodynamic stabilization of a protein by interaction with a small-molecule ligand. With increasing temperature, proteins undergo thermal denaturation resulting in irreversible aggregation and precipitation. The binding of a small molecule to its target can enhance protein stability resulting in an increased temperature of aggregation (T_agg). This distinct increase in the temperature of aggregation known as a thermal shift can identify a compound-target protein interaction in high-throughput assays or, validate a predicted interaction.