Abstract
Total internal reflection fluorescence (TIRF) microscopy is a powerful technique for interrogating protein dynamics in the membranes of living single cells. Receptor–ligand interactions are of particular interest for improving our understanding of cell signaling networks in a variety of applications. Here, we describe methods for fluorescently labeling individual receptors and their ligands, conducting single-molecule TIRF microscopy of receptors and ligands in single, living cells, and importantly, performing image analysis on the resulting time sequence of images to extract quantitative dynamics. While we use Toll-like receptor 4 and its ligand lipopolysaccharide as a specific example, the methods are general and readily extendable to other receptor–ligand systems of importance in cellular biology.
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Acknowledgments
This study was supported in part by the National Institutes of Health Director’s New Innovator Award Program, 1-DP2-OD006673-01 (probe development and experiments), as well as the Department of Energy’s Laboratory Directed Research and Development (LDRD) program (compilation and writing). Sandia National Laboratories is a multi-program laboratory managed and operated by Sandia Corporation, a wholly owned subsidiary of Lockheed Martin Corporation, for the US Department of Energy’s National Nuclear Security Administration under contract DE-AC04-94AL85000.
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Anthony, S., Carroll-Portillo, A., Timlin, J. (2015). Dynamics and Interactions of Individual Proteins in the Membrane of Single, Living Cells. In: Singh, A., Chandrasekaran, A. (eds) Single Cell Protein Analysis. Methods in Molecular Biology, vol 1346. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2987-0_13
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DOI: https://doi.org/10.1007/978-1-4939-2987-0_13
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