= Thermal shift assay
A <thermal shift assay> compares a <protein>'s temperature-dependent unfolding transition between experimental conditions, often with and without a candidate <ligand>. The measured shift reports altered thermal behaviour. It is commonly used to investigate <ligand binding> without first requiring an assay of the <protein>'s biological activity.<High-density miniaturized thermal shift assays as a general strategy for drug discovery by M. W. Pantoliano and others>[2001, Abstract; energetic coupling and miniaturized screening]{ref}
In <differential scanning fluorimetry>, an environment-sensitive fluorescent dye reports changes during heating. A temperature-controlled instrument measures <fluorescence> from matched control and compound-containing samples. The resulting <apparent protein unfolding midpoint> is a feature of the measured transition; it need not be a reversible equilibrium melting temperature.<Evaluation of fluorescence-based thermal shift assays for hit identification in drug discovery by Mei-Chu Lo and others>[2004, Abstract; dye-based unfolding measurement and instrument]{ref}
For an invented example, suppose a consistently defined midpoint changes from $48\,{}^{\circ}\mathrm{C}$ in the control to $52\,{}^{\circ}\mathrm{C}$ with a compound. Then
$$
\Delta T_m=T_{m,\mathrm{compound}}-T_{m,\mathrm{control}}=4\,\mathrm{K}.
$$
This indicates thermal stabilization under the tested conditions. It does not by itself give a room-temperature <dissociation constant> or establish inhibition of <enzyme> activity. The 2004 study compared the method with <isothermal titration calorimetry> and activity assays, illustrating the value of an independent readout.<Evaluation of fluorescence-based thermal shift assays for hit identification in drug discovery by Mei-Chu Lo and others>[Abstract; orthogonal affinity and enzymatic-assay comparisons]{ref}
<Protein denaturation>, <protein aggregation> and cleavage of <peptide bonds> are different events. A fluorescence trace alone must not be assumed to identify all three.
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