UNIT 12.7 Detecting Protein–Protein Interactions with CFP-YFP FRET by Acceptor Photobleaching

  1. Tatiana Karpova,
  2. James G. McNally

Published Online: 1 FEB 2006

DOI: 10.1002/0471142956.cy1207s35

Current Protocols in Cytometry

Current Protocols in Cytometry

How to Cite

Karpova, T. and McNally, J. G. 2006. Detecting Protein–Protein Interactions with CFP-YFP FRET by Acceptor Photobleaching. Current Protocols in Cytometry. 35:12.7:12.7.1–12.7.11.

Author Information

  1. National Institutes of Health, Bethesda, Maryland

Publication History

  1. Published Online: 1 FEB 2006
  2. Published Print: JAN 2006


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


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