FAP-tags® GPCR Trafficking Assays utilize two different fluorogens that are activated by the same FAP to detect and quantitate surface bound and internalized receptor in response to ligand binding. Both fluorogens are cell impermeable, with the βGREEN fluorogen having a higher affinity for FAP versus βRED.
GPCR Trafficking Assay (basic protocol)
- Add test compound plus βRED fluorogen
- Allow time for response
- Add βGREEN fluorogen (chase surface-bound βRED fluorogen)
- Read
Red fluorescent staining will correspond to FAP-tagged protein that was internalized and not yet returned to the cell surface. Green fluorescence represents FAP-tagged protein that either stayed at the surface or returned to the surface after receptor resensitization.
The left panel shows CXCR4 at the surface before the addition of agonist. The middle panel shows the image 20 minutes after the addition of agonist, showing receptor internalization. The final panel shows the image after the addition of the chase βGREEN fluorogen. In this image red fluorescence represents internalized FAP-tagged protein and green fluorescence represents FAP-tagged protein on the surface.
Assay Features / Benefits
- Measures real-time receptor response in living cells
- Directed to the receptor itself, not to downstream events
- Reveals dose-response, kinetics, and ligand selectivity
- Simple setup with no wash or cleanup steps
- Fluorescence is excited at a single wavelength and read at two well-separated wavelengths
- Does not require high resolution images
- Readily automated for high throughput applications
- Can be applied to any receptor