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Determination of G-protein-coupled receptor oligomerization by molecular brightness analyses in single cells

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Item Type:Article
Title:Determination of G-protein-coupled receptor oligomerization by molecular brightness analyses in single cells
Creators Name:Işbilir, A. and Serfling, R. and Möller, J. and Thomas, R. and De Faveri, C. and Zabel, U. and Scarselli, M. and Beck-Sickinger, A.G. and Bock, A. and Coin, I. and Lohse, M.J. and Annibale, P.
Abstract:Oligomerization of membrane proteins has received intense research interest because of their importance in cellular signaling and the large pharmacological and clinical potential this offers. Fluorescence imaging methods are emerging as a valid tool to quantify membrane protein oligomerization at high spatial and temporal resolution. Here, we provide a detailed protocol for an image-based method to determine the number and oligomerization state of fluorescently labeled prototypical G-protein-coupled receptors (GPCRs) on the basis of small out-of-equilibrium fluctuations in fluorescence (i.e., molecular brightness) in single cells. The protocol provides a step-by-step procedure that includes instructions for (i) a flexible labeling strategy for the protein of interest (using fluorescent proteins, small self-labeling tags or bio-orthogonal labeling) and the appropriate controls, (ii) performing temporal and spatial brightness image acquisition on a confocal microscope and (iii) analyzing and interpreting the data, excluding clusters and intensity hot-spots commonly observed in receptor distributions. Although specifically tailored for GPCRs, this protocol can be applied to diverse classes of membrane proteins of interest. The complete protocol can be implemented in 1 month.
Keywords:Confocal Microscopy, Fluorescence, Fluorescence Microscopy, Fluorescence Spectrometry, G-Protein-Coupled Receptors, HEK293 Cells, Membrane Proteins, Optical Imaging, Protein Multimerization, Signal Transduction, Single-Cell Analysis
Source:Nature Protocols
Publisher:Nature Publishing Group
Page Range:1419-1451
Date:March 2021
Additional Information:Copyright © 2021, The Author(s), under exclusive licence to Springer Nature Limited
Official Publication:https://doi.org/10.1038/s41596-020-00458-1
PubMed:View item in PubMed

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