Detecting Protein–Protein Interactions with CFP‐YFP FRET by Acceptor Photobleaching

Tatiana Karpova1, James G. McNally1

1 National Institutes of Health, Bethesda, Maryland
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 12.7
DOI:  10.1002/0471142956.cy1207s35
Online Posting Date:  February, 2006
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FRET is a light microscopy method for detecting protein‐protein interactions within intact cells. The FRET protocol presented here is for CFP‐ and YFP‐tagged proteins examined with an argon laser on a scanning confocal microscope. FRET is assayed by one of the most straightforward approaches available, namely, acceptor photobleaching. In this procedure, the YFP‐tagged protein (the FRET “acceptor”) is photobleached at a cellular site of interest, and then the intensity of the CFP‐tagged protein (the FRET “donor”) at that same site is measured. In principle, FRET is detected when the CFP intensity increases after the photobleaching of YFP. This unit describes the appropriate steps to perform this measurement, as well as the necessary controls to ensure that an increase in CFP intensity, if detected, in fact reflects bona fide FRET. Successful application of the protocol will support the conclusion that the CFP‐ and YFP‐tagged proteins directly interact at the site of the photobleaching

Keywords: Förster resonance energy transfer; FRET; acceptor photobleaching; CFP; YFP; protein interactions; LSM510 Zeiss confocal microscope; confocal microscopy; argon laser

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Table of Contents

  • Basic Protocol 1: Fret for Fixed Cells
  • Alternate Protocol 1: FRET for Live Cells
  • Commentary
  • Literature Cited
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Basic Protocol 1: Fret for Fixed Cells

  • Plasmids (Ausubel et al., ) for Protein1‐YFP and Protein2‐CFP (to test for FRET between Protein1 and Protein2) and for CFP‐YFP fusion, unconjugated CFP, and unconjugated YFP (as controls)
  • Cell line of interest
  • 2% (v/v) formaldehyde in PBS
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • Mounting medium without antifade
  • No. 1.5 coverslips
  • 6‐well tissue‐culture plate
  • Laser scanning confocal microscope: e.g., Zeiss LSM510 or similar system with:
    • Argon laser tuned to 458‐nm and 514‐nm laser lines
    • 470‐ to 500‐nm band‐pass emission filter (BP470‐500) for CFP
    • 530‐nm long‐pass emission filter (LP530) for YFP
    • Chroma q500Ip (505 nm) beam splitter
    • LSM510 physiology software (Zeiss) or comparable software enabling quantification of intensities
  • Additional reagents and equipment for transfection (Seidman et al., ) and culture ( appendix 3B) of mammalian cells

Alternate Protocol 1: FRET for Live Cells

  • Environmental chamber or a stage heater, such as a Nevtek ASI400
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Literature Cited

Literature Cited
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