Imaging Protein‐Protein Interactions by Förster Resonance Energy Transfer (FRET) Microscopy in Live Cells

Joseph A. Brzostowski1, Tobias Meckel1, Jiang Hong1, Alice Chen1, Tian Jin1

1 NIH/NIAID—LIG Imaging Facility, Rockville, Maryland
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 19.5
DOI:  10.1002/0471140864.ps1905s56
Online Posting Date:  April, 2009
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This unit describes an acceptor‐sensitized emission FRET method using a confocal microscope for image acquisition. In contrast to acceptor photobleaching, which is an end‐point assay that destroys the acceptor fluorophore, the sensitized emission method is amenable for FRET measurements in live cells and can be used to measure changes in FRET efficiency over time. The purpose of this unit is to provide a basic starting point for understanding and performing the sensitized emission method with a simple teaching tool for live‐cell imaging. References that discuss the vagaries of acquiring and analyzing FRET between individually tagged molecules are provided. Curr. Protoc. Protein Sci. 56:19.5.1‐19.5.12. © 2009 by John Wiley & Sons, Inc.

Keywords: FRET; sensitized emission; acceptor photobleaching; Zeiss LSM 510; N‐FRET; Cerulean; Venus

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Table of Contents

  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1:

  • HEK 293 (ATCC # CRL‐1573)
  • Dulbecco's modified Eagle's medium (DMEM; Invitrogen)
  • Fetal bovine serum (FBS; HyClone)
  • Phosphate buffered saline with (PBS++) and without (PBS) Ca2+ and Mg2+ (Invitrogen)
  • DNA plasmid constructs (pC5V, pC5A, pC17V, pC17A, pC32V, pC32A, and pV1)
  • Plasmid DNA isolation kit (Qiagen plasmid mini kit, Qiagen)
  • DNA transfection kit (Lipofectamine 2000, Invitrogen)
  • G418 (Sigma‐Aldrich)
  • Kanamycin (Sigma‐Aldrich)
  • Cells of interest
  • Untransfected cells to assess background autofluorescence
  • 25‐cm2 tissue culture dishes
  • 37°C, 5% CO 2 incubator
  • 6‐well culture plates (Nunc, Thermo Fisher Scientific)
  • Chambers for live‐cell imaging, e.g., coverglass‐bottomed Lab‐Tek II multi‐well chambers (Nunc); 35‐mm coverglass‐bottomed dishes (MatTek); or FCS2 environmental chamber (Bioptechs)
  • Zeiss LSM 510 META laser scanning confocal microscope or equivalent
  • Microscope environmental chamber to regulate temperature and CO 2
NOTE: CFP and YFP variants comprise the most widely used FRET pair in live cells. Alternative fluorophore considerations are discussed in Periasamy and Day ( ), Shaner et al. ( ), and Giepmans et al. ( ).
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Literature Cited

Literature Cited
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   Förster, T. 1949. Experimental and theoretical investigation of the intermolecular transfer of electronic excitation energy. Z. Naturforsch. A 4:7.
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