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Probing 2-dimensional protein–protein interactions on model membranes

Nature Protocols volume 1, pages 2091–2103 (2006)Cite this article

Abstract

This protocol describes an in vitro approach for measuring the kinetics and affinities of interactions between membrane-anchored proteins. This method is particularly established for dissecting the interaction dynamics of cytokines with their receptor subunits. For this purpose, the receptor subunits are tethered in an orientated manner onto solid-supported lipid bilayers by using multivalent chelator lipids. Interaction between the ligand with the receptor subunits was probed by a combination of surface-sensitive spectroscopic detection techniques. Label-free detection by reflectance interferometry is used for following assembly of the membrane and tethering of the receptor subunits in quantitative terms. Total internal reflection spectroscopy is used for monitoring ligand binding to the membrane-anchored receptor, for monitoring ligand-receptor interactions by FRET and for monitoring ligand-exchange kinetics. These assays can be used for determining the affinities and stabilities of ligand-receptor complexes in plane of the membrane. The techniques described in this protocol can be established in 2–3 months.

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Figure 1: Orientated and stable tethering of proteins to membranes.
Figure 2: Experimental set-up for simultaneous total internal reflection fluorescence spectroscopy-reflectance interferometry (TIRFS-Rif) detection.
Figure 3: 2-dimensional dynamics of ternary ligand-receptor complexes.
Figure 4: Detailed drawing of the flow cell and the coupling from the fiber onto the transducer.
Figure 5: Membrane assembling, conditioning and binding assay.
Figure 6: Surface concentration-dependent ligand dissociation kinetics.
Figure 8: Receptor chasing assay.
Figure 7: Ligand chasing assay.

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Authors and Affiliations

  1. Institute of Biochemistry, Biocenter N210, Johann Wolfgang Goethe-University, Max-von-Laue Straße 9, Frankfurt am Main, 60438, Germany

    Martynas Gavutis, Suman Lata & Jacob Piehler

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  1. Martynas Gavutis
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  2. Suman Lata
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  3. Jacob Piehler
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Correspondence to Jacob Piehler.

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Gavutis, M., Lata, S. & Piehler, J. Probing 2-dimensional protein–protein interactions on model membranes. Nat Protoc 1, 2091–2103 (2006). https://doi.org/10.1038/nprot.2006.270

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