UNIT 9.43 Real-Time Detection of Protein Trafficking with High-Throughput Flow Cytometry (HTFC) and Fluorogen-Activating Protein (FAP) Base Biosensor
Published Online: 2 JAN 2014
Copyright © 2001 John Wiley & Sons, Inc. All rights reserved.
Lab Protocol Title
Current Protocols in Cytometry
How to Cite
Wu, Y., Tapia, P. H., Jarvik, J., Waggoner, A. S. and Sklar, L. A. 2014. Real-Time Detection of Protein Trafficking with High-Throughput Flow Cytometry (HTFC) and Fluorogen-Activating Protein (FAP) Base Biosensor. Current Protocols in Cytometry. 67:9.43:9.43.1–9.43.11.
- Published Online: 2 JAN 2014
We combined fluorogen-activating protein (FAP) technology with high-throughput flow cytometry to detect real-time protein trafficking to and from the plasma membrane in living cells. The hybrid platform allows drug discovery for trafficking receptors, such as G protein–coupled receptors, receptor tyrosine kinases, and ion channels, which were previously not suitable for high-throughput screening by flow cytometry. The system has been validated using the β2-adrenergic receptor (β2AR) system and extended to other GPCRs. When a chemical library containing ∼1200 off-patent drugs was screened against cells expressing FAP-tagged β2AR, all known β2AR active ligands in the library were successfully identified, together with a few compounds that were later confirmed to regulate receptor internalization in a nontraditional manner. The unexpected discovery of new ligands by this approach indicates the potential of using this protocol for GPCR de-orphanization. In addition, screens of multiplexed targets promise improved efficiency with minor protocol modification. Curr. Protoc. Cytom. 67:9.43.1-9.43.11. © 2014 by John Wiley & Sons, Inc.
- high-throughput flow cytometer;
- fluorogen-activating protein;
- G protein–coupled receptor;
- receptor trafficking;
- live-cell assay