Abstract
Recent progress in super-resolution imaging (SRI) has created a strong need to improve protein labeling with probes of small size that minimize the target-to-label distance, increase labeling density, and efficiently penetrate thick biological tissues. This protocol describes a method for labeling genetically modified proteins incorporating a small biotin acceptor peptide with a 3-nm fluorescent probe, monomeric streptavidin. We show how to express, purify, and conjugate the probe to organic dyes with different fluorescent properties, and how to label selectively biotinylated membrane proteins for SRI techniques (point accumulation in nanoscale topography (PAINT), stimulated emission depletion (STED), stochastic optical reconstruction microscopy (STORM)). This method is complementary to the previously described anti-GFP-nanobody/SNAP-tag strategies, with the main advantage being that it requires only a short 15-amino-acid tag, and can thus be used with proteins resistant to fusion with large tags and for multicolor imaging. The protocol requires standard molecular biology/biochemistry equipment, making it easily accessible for laboratories with only basic skills in cell biology and biochemistry. The production/purification/conjugation steps take ∼5 d, and labeling takes a few minutes to an hour.
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Acknowledgements
We thank D. Perrais (Interdisciplinary Institute for Neuroscience, University of Bordeaux (IINS)), A. Ting (Stanford University), and S. Park (Buffalo University) for the generous gift of DNA plasmids; M. Letellier (IINS) for the slice cultures; Z. Karatas, B. Tessier, S. Antoine, and I. Gauthereau for molecular biology and biochemistry; and C. Poujol and the Bordeaux Imaging Center for providing access and support to confocal and STED setups. This work received funding from the Centre National de la Recherche Scientifique, Agence Nationale pour la Recherche (grants Synapse-2Dt and Nanodom (O.T.), Integractome and FastNano (O.R., G.G.), and SynAdh (O.T., M.S.), Conseil Régional Aquitaine, Fondation pour la Recherche Médicale, the National Infrastructure France BioImaging (grant ANR-10INBS-04-01), and the Labex BRAIN.
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I.C., M.S., and O.T. designed research and coordinated the research project. O.R. and G.G. provided their expertise and tools for the integrin model and contributed to related experiments. I.C. and M.S. performed experiments and analysis, and wrote the article. All authors discussed the results and commented on the manuscript.
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Chamma, I., Rossier, O., Giannone, G. et al. Optimized labeling of membrane proteins for applications to super-resolution imaging in confined cellular environments using monomeric streptavidin. Nat Protoc 12, 748–763 (2017). https://doi.org/10.1038/nprot.2017.010
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DOI: https://doi.org/10.1038/nprot.2017.010
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