Dose Response Curve

For Binding Affinity analysis, ligand-dependent changes in TRIC are plotted as Fnorm values vs. ligand concentration (see Concentrations).

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

In some instances, it is possible that the ligand induces a change in initial steady-state fluorescence (see Ligand-Induced Fluorescence Change). This can impact the Fnorm data but is accounted for in the Kd 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. The TRIC response of a given target is usually 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). The ‘direction’ of the curve therefore depends on the specific properties of the interacting molecules as well as the nature and the position of the fluorophore used and does not influence the result of affinity quantification.

An important parameter to judge 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.

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