Regarding fluorescence intensity or anisotropy titrations: I have noticed in the literature that in order to determine Kd of a protein-ligand complex, some people are doing titrations by recording fluorescence quenching or anisotropy using serial dilution of equimolar protein-ligand samples and NOT by adding increasing amounts of ligand to a constant concn of protein or vice versa. Is that a valid alternative to the more orthodox way of titrating a complex, can lead to an estimation of kd with the usual non-linear regression analysis and what are the advantages, if any of this approach?

Thanks