Target: To achieve a good resolution of the binding event, the concentration of the fluorescent Target needs to be lower or in the range of the Kd of the interaction. A higher target concentration will lead to a changed binding behavior in the sample, reflected in a modified Dose Response Curve shape. This, in turn, does not allow a reliable determination of the K_d. As long as the target concentration is well below the Kd, the exact target concentration does not need to be known; it can be estimated, for example, from the protein labeling and dilution procedure.

Ligand: To achieve full coverage of the binding event, the highest concentration of Ligand in the experiment should be 50-fold above the K_d of the interaction to reach Saturation. The highest ligand concentrations only 10-fold above the estimated K_d can also yield acceptable results, but higher concentrations are recommended for robust assays and high data quality. In contrast to the target, the ligand concentration must be known precisely since it directly influences the derived K_d value. In single-dose screening, the ligand concentration is typically chosen to be 10 µM for small molecules or 500 µM for fragments to be able to identify weak binders that can be improved during lead optimization. The solubility of a ligand does not only determine the weakest affinity that is possible to be measured but also the data quality. Ligand precipitation can result in Aggregation of the target molecule or even cloud the Well bottom, thereby obstructing the measurement.