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  Interrogating surface versus intracellular transmembrane receptor populations using cell-impermeable SNAP-tag substrates

Poc, P., Gutzeit, V. A., Ast, J., Lee, J., Jones, B. J., D´Este, E., et al. (2020). Interrogating surface versus intracellular transmembrane receptor populations using cell-impermeable SNAP-tag substrates. Chemical Science, 2020(30), 7871-7883. doi:10.1101/2020.01.29.924829.

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 Creators:
Poc, Pascal1, Author              
Gutzeit, Vanessa A., Author
Ast, Julia, Author
Lee, Joon, Author
Jones, Ben J., Author
D´Este, Elisa2, Author              
Mathes, Bettina1, Author              
Hodson, David J., Author
Levitz, Joshua, Author
Broichhagen, Johannes3, Author              
Affiliations:
1Max Planck Institute for Medical Research, Max Planck Society, ou_1125545              
2Department of NanoBiophotonics, MPI for Biophysical Chemistry, Max Planck Society, ou_578627              
3Chemical Biology, Max Planck Institute for Medical Research, Max Planck Society, ou_2364732              

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 Abstract: Employing self-labelling protein tags for the attachment of fluorescent dyes has become a routine and powerful technique in optical microscopy to visualize and track fused proteins. However, membrane permeability of the dyes and the associated background signals can interfere with the analysis of extracellular labeling sites. Here we describe a novel approach to improve extracellular labeling by functionalizing the SNAP-tag substrate benzyl guanine (“BG”) with a charged sulfonate (“SBG”). This chemical manipulation improves solubility, reduces non-specific staining and renders the bioconjugation handle impermeable while leaving its cargo untouched. We report SBG-conjugated fluorophores across the visible spectrum, which cleanly label SNAP-fused proteins in the plasma membrane of living cells. We demonstrate the utility of SBG-conjugated fluorophores to interrogate class A, B and C G protein-coupled receptors (GPCRs) using a range of imaging approaches including nanoscopic super-resolution imaging, analysis of GPCR trafficking from intra- and extracellular pools, in vivo labelling in mouse brain and analysis of receptor stoichiometry using single molecule pull down.

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Language(s): eng - English
 Dates: 2020-05-152020-07-022020-01-302020-08-14
 Publication Status: Published in print
 Pages: 13
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 Table of Contents: -
 Rev. Type: Peer
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Title: Chemical Science
  Other : Chem. Sci.
Source Genre: Journal
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Publ. Info: Cambridge, UK : Royal Society of Chemistry
Pages: - Volume / Issue: 2020 (30) Sequence Number: - Start / End Page: 7871 - 7883 Identifier: ISSN: 2041-6520
CoNE: https://pure.mpg.de/cone/journals/resource/2041-6520