Application note: NanoBRET^TM assay for monitoring of ligand binding to GPCRs in live cells, using the CLARIOstar^® and the PHERAstar^® FS

Due to the crucial role of G protein-coupled receptors (GPCRs) in mediating cellular responses to external stimuli, these receptors have been and will remain a prime focus for medical research and the pharmaceutical industry for many years to come. Their importance is especially highlighted by the statistic that 30-50% of marketed pharmaceuticals target GPCRs. This application note describes the first use of BRET (Bioluminescence Resonance Energy Transfer) for monitoring ligand binding to GPCRs in live cells using the NanoLuc luciferase….

A number of assay techniques are currently used to study ligand binding to GPCRs in living cells. The most well-known being the use of radioactively labelled ligands which unfortunately, is labour intensive, needs dedicated equipment and has disposal and safety issues associated with its use. More recently FRET (Fluorescence Resonance Energy Transfer), and Time Resolved-FRET techniques have also been used. However, fluorescence techniques require excitation with a high energy lamp and as cells display substantial auto-fluorescence this often leads to high background signal in the data. TR-FRET addresses this issue, but requires chemically based lanthanide labelling of receptors.

BRET requires only the addition of a suitable substrate to generate light/energy which then excites the fluorescent binding partner and donor luciferase enzyme can be expressed on the receptor of interest using an appropriate transfected cell line.