I am performing a TSA to investigate binding of a ligand to the protein by performing a serial dilution of ligand against same conc. of protein (~2uM). Interestingly at low ligand conc the fluorescence change is marginal but an obvious negative melt peak is observed at high ligand conc., but change in Tm is not significant.

To my understanding the fluorescence signal should be more or less the same with only Tm changing if there is binding. So what is the cause of fluorescence change here?

Thanks in advance!