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Investigation of a Novel, High-throughput Method for Real-time Measurement of β-arrestin Recruitment using a Live-cell Luminescence Based Assay SystemTélécharger
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February 06, 2019
Authors: P. Brescia, P. Banks, BioTek Instruments, Inc., Winooski, VT, USA; A Paguio, B Binkowski and A. Landreman, Promega Corporation, Madison, WI, USA
GPCRs and their involvement in cellular signaling remain a focal point of concerted efforts into the investigation of druggable targets. Investigation of protein: protein interactions (PPIs) involved in GPCR signaling, such as the GPCR:β-arrestin2 interaction, provides a means to better understand pathways involved with a variety of diseases such as cancer. The ability to perform live-cell measurements of these interactions remove many of the limitations of other approaches by providing real-time, kinetic results. The development of the NanoLuc® Binary Technology (NanoBiT®) in combination with Nano-Glo® Live Cell Reagent, allows realtime measurement of PPI dynamics in living cells using a simple bioluminescent readout. Here we show the ability to perform a GPCR: β-arrestin2 recruitment assay in a high-throughput, 1536-well microplate format using automated liquid handling. The NanoBiT CX3CR1/ARRB2 stable cell line was used to demonstrate this PPI when activated by addition of CX3CL1 ligand (fractalkine). A dose response titration of CX3CL1, as well as Z’-factor determination to assess assay performance, are demonstrated here.