Single-molecule microscopy of molecules tagged with GFP or RFP derivatives in mammalian cells using nanobody binders
| Autor: | Evgenia Platonova, Helge Ewers, Jan Faix, David Albrecht, Alexander Junemann, Christian M. Winterflood |
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| Rok vydání: | 2015 |
| Předmět: |
Green Fluorescent Proteins
STORM Fluorescent Antibody Technique Nanotechnology VHH PALM General Biochemistry Genetics and Molecular Biology Green fluorescent protein Microscopy Image Processing Computer-Assisted Molecule Animals Humans Single-molecule localization microscopy (SMLM) Spectral-demixing direct stochastic reconstruction microscopy (SD-dSTORM) Single particle tracking (SPT) Nanobody Single domain antibody Molecular Biology Cells Cultured Fluorescent Dyes Staining and Labeling Chemistry Biochemistry Genetics and Molecular Biology(all) Resolution (electron density) Single-Domain Antibodies Fluorescence Molecular Imaging Rats Luminescent Proteins Single-domain antibody Single Molecule Microscopy Microscopy Fluorescence Single-particle tracking |
| Zdroj: | Methods, 88 |
| ISSN: | 1046-2023 |
| DOI: | 10.1016/j.ymeth.2015.06.018 |
| Popis: | With the recent development of single-molecule localization-based superresolution microscopy, the imaging of cellular structures at a resolution below the diffraction-limit of light has become a widespread technique. While single fluorescent molecules can be resolved in the nanometer range, the delivery of these molecules to the authentic structure in the cell via traditional antibody-mediated techniques can add substantial error due to the size of the antibodies. Accurate and quantitative labeling of cellular molecules has thus become one of the bottlenecks in the race for highest resolution of target structures. Here we illustrate in detail how to use small, high affinity nanobody binders against GFP and RFP family proteins for highly generic labeling of fusion constructs with bright organic dyes. We provide detailed protocols and examples for their application in superresolution imaging and single particle tracking and demonstrate advantages over conventional labeling approaches. Methods, 88 |
| Databáze: | OpenAIRE |
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