ArticleBiophysical journal2014
Evaluation of fluorophores to label SNAP-tag fused proteins for multicolor single-molecule tracking microscopy in live cells.
Article in Biophysical journal, 2014. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 48 papers.
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Who cites it
48 citing papers in PubMed, 110 citations in OpenAlex.
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- Long-Term Single-Molecule Tracking in Living Cells using Weak-Affinity Protein Labeling.Angewandte Chemie (International ed. in English) · 2025Article
- Real-time imaging of axonal membrane protein life cycles.Nature protocols · 2024Review
- ProDOL: a general method to determine the degree of labeling for staining optimization and molecular counting.Nature methods · 2024Article
- Variable-Angle Epifluorescence Microscopy for Single-Particle Tracking in the Plant ER.Methods in molecular biology (Clifton, N.J.) · 2024Article
- Site-specific protein conjugates incorporating Para-Azido-L-Phenylalanine for cellular and in vivo imaging.Methods (San Diego, Calif.) · 2023Article
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- Dual-color DNA-PAINT single-particle tracking enables extended studies of membrane protein interactions.Nature communications · 2023Article
- Single-Molecule Displacement Mapping Indicates Unhindered Intracellular Diffusion of Small (≲1 kDa) Solutes.Journal of the American Chemical Society · 2023Article
- Harnessing Split-Inteins as a Tool for the Selective Modification of Surface Receptors in Live Cells.Chembiochem : a European journal of chemical biology · 2023Article
- Molecular sensors reveal the mechano-chemical response ofCell surface (Amsterdam, Netherlands) · 2022Article
- Choosing the Probe for Single-Molecule Fluorescence Microscopy.International journal of molecular sciences · 2022Review
- Practical observations on the use of fluorescent reporter systems in Clostridioides difficile.Antonie van Leeuwenhoek · 2022Article
- Fluorescence Imaging of Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitor Resistance in Non-Small Cell Lung Cancer.Cancers · 2022Review
- Editorial: Molecular Insights Into Ligand-Receptor Interactions on the Cell Surface.Frontiers in molecular biosciences · 2022Article
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Authors and funding
7 authors at 4 institutions in 3 countries.
Funding
No grant is acknowledged in the PubMed record.
Abstract
Single-molecule tracking has become a widely used technique for studying protein dynamics and their organization in the complex environment of the cell. In particular, the spatiotemporal distribution of membrane receptors is an active field of study due to its putative role in the regulation of signal transduction. The SNAP-tag is an intrinsically monovalent and highly specific genetic tag for attaching a fluorescent label to a protein of interest. Little information is currently available on the choice of optimal fluorescent dyes for single-molecule microscopy utilizing the SNAP-tag labeling system. We surveyed 6 green and 16 red excitable dyes for their suitability in single-molecule microscopy of SNAP-tag fusion proteins in live cells. We determined the nonspecific binding levels and photostability of these dye conjugates when bound to a SNAP-tag fused membrane protein in live cells. We found that only a limited subset of the dyes tested is suitable for single-molecule tracking microscopy. The results show that a careful choice of the dye to conjugate to the SNAP-substrate to label SNAP-tag fusion proteins is very important, as many dyes suffer from either rapid photobleaching or high nonspecific staining. These characteristics appear to be unpredictable, which motivated the need to perform the systematic survey presented here. We have developed a protocol for evaluating the best dyes, and for the conditions that we evaluated, we find that Dy 549 and CF 640 are the best choices tested for single-molecule tracking. Using an optimal dye pair, we also demonstrate the possibility of dual-color single-molecule imaging of SNAP-tag fusion proteins. This survey provides an overview of the photophysical and imaging properties of a range of SNAP-tag fluorescent substrates, enabling the selection of optimal dyes and conditions for single-molecule imaging of SNAP-tagged fusion proteins in eukaryotic cell lines.
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