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Elife. 2016 Dec 9;5. pii: e20378. doi: 10.7554/eLife.20378.

Labeling proteins inside living cells using external fluorophores for microscopy.

Teng KW1,2, Ishitsuka Y2,3, Ren P1,2, Youn Y1,2, Deng X4, Ge P3, Lee SH2,3, Belmont AS1,4, Selvin PR1,2,3,4.

Author information

1
Center for Biophysics and Quantitative Biology, University of Illinois at Urbana-Champaign, Urbana, United States.
2
Center for Physics of Living Cell, University of Illinois at Urbana-Champaign, Urbana, United States.
3
Department of Physics, University of Illinois at Urbana-Champaign, Urbana, United States.
4
Department of Cell and Developmental Biology, University of Illinois at Urbana-Champaign, Urbana, United States.

Abstract

Site-specific fluorescent labeling of proteins inside live mammalian cells has been achieved by employing Streptolysin O, a bacterial enzyme which forms temporary pores in the membrane and allows delivery of virtually any fluorescent probes, ranging from labeled IgG's to small ligands, with high efficiency (>85% of cells). The whole process, including recovery, takes 30 min, and the cell is ready to be imaged immediately. A variety of cell viability tests were performed after treatment with SLO to ensure that the cells have intact membranes, are able to divide, respond normally to signaling molecules, and maintains healthy organelle morphology. When combined with Oxyrase, a cell-friendly photostabilizer, a ~20x improvement in fluorescence photostability is achieved. By adding in glutathione, fluorophores are made to blink, enabling super-resolution fluorescence with 20-30 nm resolution over a long time (~30 min) under continuous illumination. Example applications in conventional and super-resolution imaging of native and transfected cells include p65 signal transduction activation, single molecule tracking of kinesin, and specific labeling of a series of nuclear and cytoplasmic protein complexes.

KEYWORDS:

Fluorescence; HaloTag; biophysics; cell biology; human; live-cell imaging; mouse; nanobody; single molecule; structural biology; super-resolution imaging

PMID:
27935478
PMCID:
PMC5148600
DOI:
10.7554/eLife.20378
[Indexed for MEDLINE]
Free PMC Article

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