TRIC uses changes in the temperature dependence of a measured fluorescence signal to qualitatively and quantitatively analyze molecular interactions. Sometimes the fluorescence signal that is measured before the IR-Laser induced temperature change (the so-called Initial Fluorescence) can also be ligand-dependent. There are two major reasons for an increase in initial fluorescence of the labeled target molecule when a ligand is added:
- The interaction between ligand and target molecule changes the chemical environment of the fluorophore leading to higher fluorescence intensity.
- The ligand itself is fluorescent in the red spectral range measured by Dianthus instruments.
Empirical investigation has revealed that initial fluorescence changes induced by an added ligand that are below a threshold of ±20 % do not significantly affect the obtained TRIC signals. In single dose measurements (see Single-Dose Screening) changes below this threshold are therefore ignored. Should the measured initial fluorescence of the target in presence of a ligand exceed +20 % the ligand is flagged as being autofluorescent. To distinguish between ligand autofluorescence and a binding- induced effect, additional control experiments are required. In affinity measurements (see Affinity Screening) initial fluorescence changes that influence the measured TRIC signals are compensated for in the Kd Fit Model (see corresponding article). It is still advisable to determine the origin of initial fluorescence changes in a control experiment.
The simplest control experiment is to measure a solution of the ligand molecule in the absence of target. If fluorescence is observed the ligand is indeed autofluorescent. If no fluorescence is observed for the ligand alone, the increase in target fluorescence is likely binding related. One option to work around an autofluorescent ligand is to reverse the setup, using the ligand as fluorescent target molecule and titrating the molecule that was initially the target, if a non-fluorescent variant of it is available.