The FRET (Fluorescence Resonance Energy Transfer) technology allows to detect distances of a few tens of angstroms between
2 fluorophores, and is therefore a technique of choice to study molecular interactions.
Here, it allows to measure the binding between a membrane receptor fused to a fluorophore and a ligand fused to another fluorophore. The binding is measured on living whole cells. The assay is performed under homogeneous conditions and does not require any washing.
The cells overexpress the receptor fused to a donor fluorophore, GFP (Green Fluorescence Protein).
A selection of plasmid constructs and cells are available at PCBIS.
The ligand is fused to a compatible acceptor fluorophore, whose excitation spectrum at least partially overlaps the emission spectrum of the donor fluorophore.
FRET is a non-radiative energy transfer between the excited state donor fluorophore and the acceptor fluorophore, when they are close enough. This results in a decrease in the light emitted by the donor fluorophore, and an increase in the light emitted by the acceptor fluorophore.
The detection of the interaction between the receptor and the ligand, which results in a decrease of the fluorescence of the donor fluorophore, is performed by a microplate reader or a spectrofluorimeter. By competition, it is possible to detect the binding of an unlabeled molecule on the receptor.
To be adapted according to the test
| Format | 96 well or 1 mL vessel | ||
| Cellular model | suspension of HEK293 Possibility to work with other cell lines | ||
Cell distribution | |||
| Baseline reading | |||
Addition of fluorescent ligand and unlabeled compound | |||
| Incubation | |||
Reading | |||
Notes :
Format : 1 mL vessel
Cells : HEK293 suspension cells overexpressing apelin receptor fused to GFP
All models can be adapted to your request.