Thermal shift assay is a microplate binding assay to quickly screen buffer/ligand conditions by monitoring thermal melting curves. The differential scanning fluorimetry (DSF) method uses an environmentally sensitive dye. The fluorescence signal increases as a function of temperature in the hydrophobic environment introduced by protein unfolding during heat-denaturation. The plot of the fluorescence signal as a function of temperature is a sigmoid curve. The inflection point is usually referred to as the melting temperature (Tm) where 50% of the protein is unfolded. An increase in Tm values will identify stabilizing conditions. Hydrophobic proteins cannot be analyzed using DSF due to high fluorescence backgrounds. With the differential static light scattering, aggregation of unfolded proteins upon heat-denaturation is monitored by light scattering. In this latter case, the inflection point of the sigmoid curve is called Tagg. Very small amounts of protein sample (1-15 µg per well) areneeded to set up a thermal shift assay.
The fluorescence signal as a function of temperature can be monitored using a variety of plate readers, including RT-PCR devices. The dye with the most favorable properties for thermal shift assay is SYPRO® orange, owing to its high signal-to-noise ratio. Analysis of thermal shift data is performed using the software provided with the screening instrument. DSLS measurements are performed using a StarGazer® instrument (Harbinger Biotechnology and Engineering Corporation) in 384-well clear bottom plates. The temperature gradient is usually performed in the range of 20–90°C with a standard ramp of 0.5°C over the course of 45 minutes.
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