Monitoring receptor ligand binding in living cells
A number of assay techniques are used to study ligand binding to GPCRs in living cells. The most well-known being the use of radioactively labelled ligands which unfortunately, is labor 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.
In this application note we will show how the BRET technique can be used to monitor receptor ligand binding. A cAMP BRET biosensor system harboring Renilla luciferase and YFP proved to be a successful approach. Forskolin dose response curves are presented. Although the intrinsic assay window is quite small, the CLARIOstar® is sensitive enough to detect small luminescence changes by either using BRET specific emission filters or by using the LVF monochromator™.