Summary
A robust method to directly measure ligand–receptor binding interactions using fluorescence cross-correlation spectroscopy (FCCS) is described. The example receptor systems demonstrated here are the human mu-opioid receptor, a representative G protein-coupled receptor (GPCR), and Streptavidin, but these general protocols can be extended for the analysis of many membrane receptors. We present methods for the preparation of GPCR-containing membrane nanopatches that appear to have the shapes of nanovesicles, labeling of proteins in membrane vesicles, in addition to the coupling of quantum dots (QDs) to peptide ligands. Further, we demonstrate that reliable binding information can be obtained from these partially purified receptors.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Cooper, M.A. (2004) Advances in membrane receptor screening and analysis. J. Mol. Recognit. 17, 286–315.
Shanmugavadivu, B., Apell, H.J., Meins, T., Zeth, K., and Kleinschmidt, J.H. (2007) Correct folding of the beta-barrel of the human membrane protein VDAC requires a lipid bilayer. J. Mol. Biol. 368, 66–78.
Powalska, E., Janosch, S., Kinne-Saffran, E., Kinne, R.K., Fontes, C.F., Mignaco, J.A., and Winter, R. (2007) Fluorescence spectroscopic studies of pressure effects on Na+, K(+)-ATPase reconstituted into phospholipid bilayers and model raft mixtures. Biochemistry 46, 1672–1683.
Killian, J.A. (1998) Hydrophobic mismatch between proteins and lipids in membranes. Biochim. Biophys. Acta. 1376, 401–415.
Swift, J.L., Burger, M.C., Massotte, D., Dahms, T.E., and Cramb, D.T. (2007) Two-photon excitation fluorescence cross-correlation assay for ligand-receptor binding: cell membrane nanopatches containing the human micro-opioid receptor. Anal. Chem. 79, 6783–6791.
Swift, J.L., Heuff, R., and Cramb, D.T. (2006) A two-photon excitation fluorescence cross-correlation assay for a model ligand-receptor binding system using quantum dots. Biophys. J. 90, 1396–1410.
Swift, J.L., and Cramb, D.T. (2008) Nanoparticles as fluorescence labels: Is size all that matters? Biophys. J. 95, 865–876.
Acknowledgments
This work was supported by the National Sciences and Engineering Council of Canada, the Alberta Ingenuity Fund, the University of Calgary, and the Canadian Institute for Photonics Innovation.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2009 Humana Press, a part of Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Swift, J.L., Burger, M.C., Cramb, D.T. (2009). A Quantum Dot-Labeled Ligand–Receptor Binding Assay for G Protein-Coupled Receptors Contained in Minimally Purified Membrane Nanopatches. In: Leifert, W. (eds) G Protein-Coupled Receptors in Drug Discovery. Methods in Molecular Biology, vol 552. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-60327-317-6_24
Download citation
DOI: https://doi.org/10.1007/978-1-60327-317-6_24
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-60327-316-9
Online ISBN: 978-1-60327-317-6
eBook Packages: Springer Protocols