Standard operating procedure for fluorescent thermal shift assay (FTSA) for determination of protein–ligand binding and protein stability

A standard operating procedure for a fluorescence-based thermal shift assay (FTSA) is provided describing its typical applications, advantages and limitations. FTSA is a simple, robust, universal and quick assay to determine protein–ligand binding affinities and protein stabilities in the presence of various excipients and solution conditions. Therefore, the assay is very useful for the straightforward characterization of new recombinantly produced proteins. The assay has a wide dynamic range enabling simultaneous determination of affinities in the milimolar to picomolar range. The assay could be used for essentially any protein that is sufficiently soluble and stable in the studied aqueous solution. Here we provide examples and typical experimental protocols for both affinity and stability determinations.