Abstract
Identification of small molecules that interact specifically with the ligand-binding domains (LBDs) of nuclear receptors (NRs) can be accomplished using a variety of methodologies. Here, we describe the use of differential scanning fluorimetry to identify these ligands, a technique that requires no modification or derivatization of either the protein or the ligand, and uses an instrument that is becoming increasingly affordable and common in modern molecular biology laboratories, the quantitative, or real-time, PCR machine. Upon being introduced to specific ligands, nuclear receptors undergo structural and dynamic changes that tend to increase molecular stability, which can be measured by the resistance of the protein to heat denaturation. Differential scanning fluorimetry (DSF) uses a dielectric sensitive fluorescent dye to measure the thermal denaturation, or "melting" point (Tm) of a protein under different conditions, in this case in the absence and presence of a candidate ligand. Using DSF, multiple candidates can be screened at once, in numbers corresponding to plate size of the instrument used (e.g., 96- or 384-well), allowing significant throughput if a modest library of compounds needs to be tested.