Interrogating surface versus intracellular transmembrane receptor populations using cell-impermeable SNAP-tag substrates.
Interrogating surface versus intracellular transmembrane receptor populations using cell-impermeable SNAP-tag substrates.
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DOI：
10.1039/d0sc02794d
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发表时间：
2020-07-07
期刊：
影响因子：
8.4
通讯作者：
Broichhagen J
中科院分区：
文献类型：
--
作者：
Poc P;Gutzeit VA;Ast J;Lee J;Jones BJ;D'Este E;Mathes B;Lehmann M;Hodson DJ;Levitz J;Broichhagen J
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 labelling sites. Here we describe a novel approach to improve extracellular labelling by functionalizing the SNAP-tag substrate benzyl guanine (“BG”) with a charged sulfonate (“SBG”). This chemical manipulation can be applied to any SNAP-tag substrate, 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 superresolution 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. Impermeable SNAP-tag substrates allow exclusive labelling of receptors on the cell membrane for nanoscopy, SiMPull and in vivo use.
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影响因子：
16.6
作者：
Jones B;Buenaventura T;Kanda N;Chabosseau P;Owen BM;Scott R;Goldin R;Angkathunyakul N;Corrêa IR Jr;Bosco D;Johnson PR;Piemonti L;Marchetti P;Shapiro AMJ;Cochran BJ;Hanyaloglu AC;Inoue A;Tan T;Rutter GA;Tomas A;Bloom SR
通讯作者：
Bloom SR
影响因子：
4
作者：
Farrants, Helen;Gutzeit, Vanessa A.;Broichhagen, Johannes
通讯作者：
Broichhagen, Johannes
影响因子：
7.7
作者：
Gutzeit, Vanessa A.;Thibado, Jordana;Levitz, Joshua
通讯作者：
Levitz, Joshua
影响因子：
48
作者：
Grimm JB;Muthusamy AK;Liang Y;Brown TA;Lemon WC;Patel R;Lu R;Macklin JJ;Keller PJ;Ji N;Lavis LD
通讯作者：
Lavis LD
DOI：
10.1038/nrd.2017.178
发表时间：
2017-12
期刊：
Nature reviews. Drug discovery
影响因子：
--
作者：
Hauser AS;Attwood MM;Rask-Andersen M;Schiöth HB;Gloriam DE
通讯作者：
Gloriam DE