For Binding Affinity analysis with Spectral Shift, when the fluorophore is attached to the target, the emission wavelength spectrum of the complex has a defined shape (full line). Upon binding to a ligand, the spectrum can be shifted to the red (dashed line) or to the blue (dotted line). Therefore, the 670 nm/650nm ratio values are plotted vs. ligand concentrations.

For Binding Affinity analysis with TRIC, ligand-dependent changes are plotted as Fnorm values vs. ligand concentration. The TRIC response of a given target is typically a reduction in fluorescence in the detection volume upon induction of a temperature gradient, represented by a descending TRIC-trace. The TRIC response of the Complex, however, can be either stronger than that of pure target (reducing the fluorescence even further), or it can be weaker (causing a smaller reduction in fluorescence).

For affinity quantification, the resulting data is approximated with one of two fit models (note that the Hill model is only available in DI.Screening Analysis and not in DI.Control):

• 1. The Kd Fit Model assumes a 1:1 interaction stoichiometry and is based on the law of mass action.
• 2. The Hill Model is a simplified model for interactions with Cooperativity and does not return the Kd but the EC50.

In some instances, it is possible that the ligand induces a change in the initial fluorescence (650 nm for Spectral Shift and 670 nm at F₀ for TRIC). This can impact the data but is accounted for in the K_d fit model.

Irrespective of the mode of action of the ligand of interest, the 'directionality' of the dose-response curve is irrelevant for further analysis. Therefore, the 'direction' of the curve depends on the specific properties of the interacting molecules and the nature and position of the fluorophore used and does not influence the result of affinity quantification.

An important parameter for judging the quality of binding affinity experiments is the signal-to-noise ratio. Signal-to-noise values higher than 12 usually correspond to excellent data quality, although a Signal-to-noise higher than 5 is acceptable.

Note: Also read the articles on Response Amplitude and Response Quality to learn more about response amplitudes and their significance.