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Fluorescent Thermal Shift Assays (FTSA)

Also known as ThermoFluor, differential scanning fluorimetry (DSF) or temperature-dependent fluorescence (TdF), FTSA exploits the phenomenon whereby the binding of a ligand enhances the thermal stability of its target protein, this phenomenon can be used to provide real-time information with respect to compound-target engagement. Although a simple technique, FTSA has the power to add valuable information at various stages of a drug discovery project.

Using an environmentally-sensitive fluorescent dye, FTSA quantifies in real-time the thermal melting of a protein. Compounds that produce a significant shift in the melting temperature of the target protein are identified as true binders. Sygnature Discovery uses a CFX384 RT-PCR System to perform these assays and our scientists are experienced in the analysis and interpretation of FTSA data.


Figure 1: Illustration of the fluorescent responses observed as a protein unfolds in a thermal shift experiment

Figure 2: Data from a thermal shift experiment assessing the binding of the p38 inhibitor SB 202190

Figure 3: Data illustrating the correlation in compound binding affinities obtained by thermal shift (FTSA) vs. SPR


  1. Redhead, M. et al. A combinatorial biophysical approach, FTSA and SPR for identifying small molecule ligands and PAINs. Anal. Biochem. 479, 63–73 (2015).
  2. Redhead, M. et al. Thermal Shift as an Entropy-Driven Effect. Biochemistry acs.biochem.7b00860 (2017).
  3. Scott, A. D., “Fluorescent thermal shift assays for identifying small molecule ligands” in Biophysical Techniques in Drug Discovery 208–238 (The Royal Society of Chemistry, 2017).

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