Journal
NATURE PROTOCOLS
Volume 12, Issue 4, Pages 748-763Publisher
NATURE PUBLISHING GROUP
DOI: 10.1038/nprot.2017.010
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Funding
- Centre National de la Recherche Scientifique
- Agence Nationale pour la Recherche
- Integractome and FastNano
- SynAdh
- Conseil Regional Aquitaine
- Fondation pour la Recherche Medicale
- National Infrastructure France BioImaging [ANR-10INBS-04-01]
- Labex BRAIN
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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 SRSRI techniques (point accumulation in nanoscale topography (PANT), 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 similar to 5 d, and labeling takes a few minutes to an hour.
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