Differential scanning fluorimetry (DSF) is a rapid and inexpensive screening method for analyzing the melting behavior of proteins1. “Melting” describes the progressive unfolding or dissociation of a protein that occurs when it is heated. DSF can be used to identify and characterize small molecule ligands that bind and stabilize proteins. For example, the P53 protein is important for cellular defense against cancer. Mutations in P53 can lower its melting temperature, render it inactive and lead to rapid denaturation. The identification of small ligand molecules that can bind to mutated sites and stabilize P53 has clear potential as a cancer therapy2. DSF monitors protein melting fluorescently using a dye with affinity for hydrophobic domains of the molecule. As a protein is melted, its hydrophobic domains are progressively exposed. This allows more dye to bind and increases the fluorescent signal emitted. The melting temperature (TM) of a protein is measured using the Rotor-Gene 6000 by continuous monitoring of the thermal unfolding of the protein in the presence of, for example, the SYPRO orange dye2. The TM of a protein is the inflection point of the melt curve generated. A simple curve-fitting procedure allows quick calculation of the transition midpoint; the difference in the temperature of this midpoint in the presence and absence of ligand is related to the binding affinity of the particular ligand molecule (which can be a low-molecular-weight compound, a peptide or a nucleic acid). An important advantage of DSF for screening small ligand molecules is that considerably less protein is required compared to alternative screening methods (approximately 0.5 nmol protein per assay) while providing a quantitative biophysical measurement. DSF can thus be applied to samples for which aggregation or low stability hinders purification. Moreover, use of the Corbett Robotics CAS-1200 dispensing robot enables large ligand libraries to be screened with high throughput. References Niesen et al 2007, The use of differential scanning fluorimetry to detect ligand interactions that promote protein stability. Nat Protoc. 2007; 2(9):2212-21 Boeckler F, Joerger A, Jaggi G, Rutherford T, Veprintsev D and Fersht A. Targeted rescue of a destabilised mutant of p53 by an in silico screened drug. PNAS 2008;105(30):10360-10365

Example data (click to enlarge):  High-resolution protein melt curves from a ligand library screen  Data was obtained using a Rotor-Gene 6000 real-time rotary analyzer   Resources Download the Protein Melt Analysis Application Note  (313kb)