title: Optimized labeling of membrane proteins for applications to super-resolution imaging in confined cellular environments using monomeric streptavidin.
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.
authors.affiliation: Interdisciplinary Institute for Neuroscience, UMR 5297, Centre National de la Recherche Scientifique, Bordeaux, France.|Interdisciplinary Institute for Neuroscience, University of Bordeaux, Bordeaux, France.
authors.forename: Ingrid
authors.initials: I
authors.lastname: Chamma
authors.affiliation: Interdisciplinary Institute for Neuroscience, UMR 5297, Centre National de la Recherche Scientifique, Bordeaux, France.|Interdisciplinary Institute for Neuroscience, University of Bordeaux, Bordeaux, France.
authors.forename: Olivier
authors.identifier: 0000-0002-6932-931X
authors.initials: O
authors.lastname: Rossier
authors.affiliation: Interdisciplinary Institute for Neuroscience, UMR 5297, Centre National de la Recherche Scientifique, Bordeaux, France.|Interdisciplinary Institute for Neuroscience, University of Bordeaux, Bordeaux, France.
authors.forename: Grégory
authors.initials: G
authors.lastname: Giannone
authors.affiliation: Interdisciplinary Institute for Neuroscience, UMR 5297, Centre National de la Recherche Scientifique, Bordeaux, France.|Interdisciplinary Institute for Neuroscience, University of Bordeaux, Bordeaux, France.
authors.forename: Olivier
authors.initials: O
authors.lastname: Thoumine
authors.affiliation: Interdisciplinary Institute for Neuroscience, UMR 5297, Centre National de la Recherche Scientifique, Bordeaux, France.|Interdisciplinary Institute for Neuroscience, University of Bordeaux, Bordeaux, France.
authors.forename: Matthieu
authors.identifier: 0000-0001-5465-5641
authors.initials: M
authors.lastname: Sainlos
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D002462:Cell Membrane / Q000378:metabolism; D006224:Cricetinae; D005456:Fluorescent Dyes / Q000737:chemistry*; D051379:Mice; D008958:Models, Molecular; D011487:Protein Conformation; D013194:Staining and Labeling / Q000379:methods*; D019809:Streptavidin / Q000737:chemistry*
chemical_list: D005456:Fluorescent Dyes; D019809:Streptavidin