Labeling proteins inside living cells using external fluorophores for microscopy

Kai Wen Teng, Yuji Ishitsuka, Pin Ren, Yeoan Youn, Xiang Deng, Pinghua Ge, Andrew S. Belmont, Paul R. Selvin

Research output: Contribution to journalArticle

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.

Original languageEnglish (US)
Article numbere20378
JournaleLife
Volume5
Issue numberDECEMBER2016
DOIs
StatePublished - Dec 9 2016

ASJC Scopus subject areas

  • Neuroscience(all)
  • Immunology and Microbiology(all)
  • Biochemistry, Genetics and Molecular Biology(all)

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  • Cite this

    Teng, K. W., Ishitsuka, Y., Ren, P., Youn, Y., Deng, X., Ge, P., Belmont, A. S., & Selvin, P. R. (2016). Labeling proteins inside living cells using external fluorophores for microscopy. eLife, 5(DECEMBER2016), [e20378]. https://doi.org/10.7554/eLife.20378