PT  - JOURNAL ARTICLE
AU  - Pascal Poc
AU  - Vanessa A. Gutzeit
AU  - Julia Ast
AU  - Joon Lee
AU  - Ben J. Jones
AU  - Elisa D’Este
AU  - Bettina Mathes
AU  - David J. Hodson
AU  - Joshua Levitz
AU  - Johannes Broichhagen
TI  - Interrogating surface versus intracellular transmembrane receptor populations using cell-impermeable SNAP-tag substrates
AID  - 10.1101/2020.01.29.924829
DP  - 2020 Jan 01
TA  - bioRxiv
PG  - 2020.01.29.924829
4099  - http://biorxiv.org/content/early/2020/01/30/2020.01.29.924829.short
4100  - http://biorxiv.org/content/early/2020/01/30/2020.01.29.924829.full
AB  - 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.