We aim to study the biochemical activity of proteins with unknown functions. Co-factor binding is not only essential for the activity of some proteins, but may also alter their thermal stability. One practical application of this phenomenon lies in utilizing the change in thermal stability as measured by the protein's melting temperature to analyze ligand binding.
Thermal shift assay is a versatile tool for investigating protein interactions with potential co-factors or drugs, and for identifying stabilizing conditions for crystallography. Some of its advantages over other screening methods are its relatively simple setup and high throughput with 96 or 384 well plates. Selenoprotein O, or SelO, is an evolutionarily conserved pseudokinase that transfers adenosine monophosphate from ATP to protein substrates in a post-translational modification known as AMPylation.
In this protocol, we use TSA to analyze nucleotide and metal binding to SelO.
