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 target concentration higher than the Kd will lead to a modified binding behavior in the sample, reflected in a modified Dose Response Curve shape. This in turn does not allow an accurate determination of the Kd . As long as the target concentration is well below the Kd the exact target concentration does not need to be known, but can be estimated, for example from the Protein Labeling and dilution procedure.
Ligand: To achieve full Saturation of the binding event, the highest concentration of Ligand in the experiment should be 50-fold above the Kd of the interaction. . Highest ligand concentrations of only 10- fold above the estimated Kd 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 needs to be known precisely since it directly influences the derived Kd value. In single-dose screening the ligand concentration is typically chosen to be in the mM range to be able to identify all binders, including Weak Binders that can then 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 bottom of the Well, thereby obstructing the measurement.