Principle of fluorescence
Fluorescence is the physical property of some molecules, called Fluorophores, to be able to absorb a photon and emit a photon of lower energy in return. Upon photon absorption, the energy level of the fluorophore changes from the ground state (S0) to an excited state (S1). From this excited state, the molecule will return spontaneously to the ground state, emitting a photon. The absorption and emission wavelengths are specific to each fluorophore.
Fluorescence is routinely used in the life sciences to monitor or track biomolecules. Only a few natural substances exhibit autofluorescence (see Ligand Autofluorescence), attaching a fluorophore to a biomolecule provides a way of specifically observing this (type of) molecule.This is also done for TRIC experiments. The fluorescence of one interaction partner, called the Target, allows the monitoring of the TRIC signal of this molecule, and how this changes upon the binding of a ligand.
In the context of TRIC, the fluorescence signal of the target needs to have a certain intensity. We recommend at least 2000 fluorescence counts for the labeled target. The ligand alone should show less than 20% of the target fluorescence counts. The buffer alone should have a fluorescence background of less than 50% of the target fluorescence.
Recommendations when fluorescence is too high:
- Reduce concentration of fluorescent Target.
- Reduce Excitation Power.
- Check Protein Labeling procedure: high fluorescence can mean that there was a mistake in sample preparation. Track whether correct protein and dye concentrations were used during labeling. We recommend a 3-fold molar excess of dye over protein, with a protein concentration of at least 2 µM.
- Check ligand and buffer auto-fluorescence (see Ligand Autofluorescence). High
fluorescence can stem from ligand or buffer autofluorescence. If buffer auto- fluorescence is high, reduce the concentration of fluorescent components if applicable. Note that some bioliquids such as serum can show autofluorescence in the red spectrum. If the ligand shows autofluorescence, perform negative controls.
Recommendations when fluorescence is too low:
- Increase concentration of fluorescent Target.
- For small discrepancies: adjust the Excitation Power of the LED. Generally, the signal is proportional to the concentration and to the excitation power. However high excitation powers can cause significant Photobleaching, which reduces data quality.
- Suppress biomolecule Adsorption.
- Low-bind tubes / non-binding MTPs have passivated surfaces which prevent biomolecule adsorption. Therefore, adsorption of fluorescent target or non-fluorescent ligand can be prevented during pipetting steps.
- Low fluorescence may be due to low efficiency in the Protein Labeling reaction.
This can have multiple causes:
- Ensure that the protein contains surface-exposed lysine / cysteine residues / that the His-tag is freely accessible.
- Check that no thiol-based Reducing Agents (DTT, β-ME) were present during labeling.
- Ensure that freshly resuspended dye in DMSO is used. Once in aqueous buffer, the dye starts hydrolyzing quickly, greatly reducing the labeling efficiency.
- Track whether correct protein and dye concentrations were used during labeling. A 3-fold molar excess of dye over protein is recommended , with a protein concentration of at least 2 µM.