Ligand-Induced Fluorescence Change
TRIC detects biomolecular interactions by following changes in target fluorescence upon binding of ligands. Ligand binding causes a change in the TRIC signal of the target, which enables binding detection. Sometimes, ligand binding directly leads to a change in Initial Fluorescence. It can manifest both, as a ligand concentration dependent fluorescence decrease, or as a ligand dependent fluorescence increase. In both cases, the impact of this initial fluorescence change on the measured Fnorm data is accounted for in the Kd Fit Model. In case of an initial fluorescence change smaller than ± 20 % of the target’s initial fluorescence, the initial fluorescence change can be ignored. In case of an initial fluorescence change higher than ± 20 % of the target’s initial fluorescence, it is necessary to perform some control experiments to rule out possible artifacts. The possible causes for an artifact and the necessary measures are:
- A ligand concentration dependent increase in initial fluorescence can be caused by Ligand Autofluorescence. If the ligand itself is fluorescent, complex fluorescence will be higher than target fluorescence alone. In this case, the assay setup has to be changed. Ligand autofluorescence can be tested by comparing the fluorescence of a buffer-filled well with that of a well containing ligand only in absence of target.
- A ligand concentration dependent decrease in initial fluorescence (i.e. Quenching)
can also be caused if the ligand absorbs light in the relevant range (inner filter effect). In that case complex fluorescence will be lower than target fluorescence. This effect is not binding-specific, so it is independent of the target molecule, and it will also happen in a sample that includes fluorophore (see Fluorophores) and Ligand, but not the Target. In order to test for this, compare the fluorescence signal of the fluorophore (dye, fusion protein) alone, without being attached to a target molecule, to a com- bination of fluorophore and ligand.
- If neither of the above cases could be confirmed as a cause for the initial fluorescence
change, fluorescence variations in both directions can also arise if the addition of the ligand to the target is influencing target aggregation or target adsorption to the reaction tubes or other labware. This means that material is lost and the resulting measurement will be unreliable. In order to rule out this possibility, please perform a Specificity Test.