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

Ali Işbilir, Robert Serfling, Jan Möller, Romy Thomas, Chiara De Faveri, Ulrike Zabel, Marco Scarselli, Annette G. Beck-Sickinger, Andreas Bock, Irene Coin, Martin J. Lohse*, Paolo Annibale

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

6 Citations (Scopus)


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.

Original languageEnglish
Pages (from-to)1419-1451
Number of pages33
JournalNature Protocols
Issue number3
Early online date29 Jan 2021
Publication statusPublished - Mar 2021


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