Thermal denaturation assays in chemical biology

Thermal denaturation-based methods are becoming increasingly used to characterize protein stability and interactions. Recent technical advances have made these methods more suitable for high throughput screening. Reasonable throughput and the ability to perform these screens using commonly used instruments, such as RT-PCR machines or simple plate readers equipped with heating devices, facilitate these experiments in almost any laboratory. Introducing an aggregation-based monitoring approach as well as alternative fluorophores has allowed the screening of a wider range of proteins, including membrane proteins, against large chemical libraries. Thermal denaturation-based methods are independent of protein function, which is especially useful for the identification of orphan protein function. Here, we review applications of thermal denaturation-based methods in characterizing protein stability and ligand binding, and also provide information on protocol modifications that may further increase throughput.