A binding affinity experiment is performed to monitor the target's signal over a dilution series of the ligand in order to derive the binding affinity (Kd). Using a wide range of ligand concentrations (typically 16 points of a 1:1 serial dilution, spanning several orders of magnitude) allows one to derive a binding curve with several data points in the baseline (unbound), transition and saturation (bound), see Figure 1 below.

Figure 1: Illustration of binding curve

Target concentration

In order to get a clear resolution of the binding event, the concentration of the fluorescent target needs to be below or in the range of the Kd of the interaction. A higher target concentration will lead to a change in binding behavior of the sample, reflected in a modified binding curve shape. This in turn does not allow a reliable extraction of the Kd. So long as the target concentration is well below the Kd, the exact concentration does not need to be known, but can be estimated, for example from the labeling and dilution procedure. We recommend starting your assay with 20 nM fluorescent target and decrease or increase the concentration subsequently, if needed.

Ligand concentration

To capture 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 may yield acceptable results; however, higher concentrations are recommended for better data quality. In contrast to the target, the ligand concentration needs to be precisely known since it directly influences the derived Kd value.

All described situations can be simulated in the Concentration Finder tool for illustration purposes. This tool is useful for planning experiments and can be found within the MO.Control software on the Plan page by clicking the pen within the ligand section next to “Highest concentration in this assay”:

In case the Kd of the interaction is unknown, start with the highest ligand concentration accessible and fully soluble in the used buffer system. Choose a broad ligand concentration series and adjust the highest ligand concentration for subsequent experiments accordingly, so that the Kd is roughly 50-fold below.

Serial dilutions

Serial dilutions of the ligand of interest are prepared and mixed with a constant concentration of the target molecule. The most common dilution series are 1:1 dilutions of either 8, 12, 16 or 24 points. Depending on the dilution factor and the number of dilutions prepared, one can cover either a broad or a narrow concentration range. The covered concentration range and the dissociation constants that can be resolved by a particular dilution can be easily simulated and visualized in the concentration finder tool in MO.Control (see above). Refer to this article for more details on serial dilutions - Ligand serial dilution – NanoTemper Technologies.