I wonder if adding pepties or nucleic acids rather than small molecules will still work. In principle it should, but I am afraid that sypro orange would bind to peptides (they might contains hydrophobic resides(, resulting in messy curves, and nucleic acids could interfere as well (don't know if sypro would bind there as well). Has everyone ever tried?
TSA will work with peptides and nucleic acids. Why not? The dye (SyproOrange, ANS or other appropriate probe) may bind to the peptide or the nucleic acid, in the same way it may bind to the protein target. The key issue is if the dye will bind more or less when the protein target unfolds upon temperature denaturation.
On the other hand, not all dyes will work with all protein targets.
Regarding Kd determination through TSA, you should be careful. The extent of the stabilization effect (increase in Tm) depends on the ligand binding affinity and the ligand concentration (as well as the protein concentration and the experimental conditions, of course). But, even if you use two ligands at the same concentrations and under the same experimental setup, the stabilization effects do not necessarily correlate with their binding affinities. Thus, two ligands with the same binding affinity at 25 degrees Celsius may induce different changes in Tm on the protein target. And two ligands with the different binding affinities at 25 degrees Celsius may induce the same change in Tm on the protein target. And this is because as the temperature increases along the TSA, the ligand binding affinity is also changing (according to the binding enthalpy and heat capacity, two parameters that may be very different between the two ligands). Therefore, TSA allows you to screen a chemical library to obtain a YES/NO answer for selecting binders, but no direct affinity ranking can be obtained. However, if you analize properly the TSA including explicitly the coupling between the conformational equilibrium and the ligand binding equilibrium, you may estimate the ligand binding affinity from TSA.
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