Application note: Monitoring receptor ligand binding in living cells
In this application note we will show how a cAMP BRET biosensor can be used to monitor ligand binding with the help of the CLARIOstar microplate reader…
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 harbouring 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.
This application note is restricted - login or subscribe free to access
Thank you for visiting our website. To access this content in full you'll need to login. It's completely free to subscribe, and in less than a minute you can continue reading. If you've already subscribed, great - just login.
Why subscribe? Join our growing community of thousands of industry professionals and gain access to:
- quarterly issues in print and/or digital format
- case studies, whitepapers, webinars and industry-leading content
- breaking news and features
- our extensive online archive of thousands of articles and years of past issues
- ...And it's all free!
Related content from this organisation
- Product hub: Targeted protein degradation – next-generation therapeutics
- ebook: Binding kinetics in drug discovery
- Application note: Competitive high-throughput analysis of kinase inhibitor binding kinetics
- Product hub: High-throughput screening of cyclic peptide libraries for developing drugs to challenging targets
- Application note: Screening for optimal association and dissociation rates of ligands using a live cell binding assay