Quantitative fluorescence resonance energy transfer (FRET) measurement with acceptor photobleaching and spectral unmixing

Authors

  • Y. GU,

    Corresponding author
    1. Light Microscopy, Cancer Research UK London Research Institute, Lincoln's Inn Fields Laboratories, London WC2A 3PX, U.K.
    Search for more papers by this author
  • W. L. DI,

    1. Light Microscopy, Cancer Research UK London Research Institute, Lincoln's Inn Fields Laboratories, London WC2A 3PX, U.K.
    2. Centre for Cutaneous Research, Institute of Cell and Molecular Science, Barts and the London School of Medicine and Dentistry, Queen Mary, University of London, London E1 2AT, U.K.
    Search for more papers by this author
  • D. P. KELSELL,

    1. Light Microscopy, Cancer Research UK London Research Institute, Lincoln's Inn Fields Laboratories, London WC2A 3PX, U.K.
    2. Centre for Cutaneous Research, Institute of Cell and Molecular Science, Barts and the London School of Medicine and Dentistry, Queen Mary, University of London, London E1 2AT, U.K.
    Search for more papers by this author
  • D. ZICHA

    1. Light Microscopy, Cancer Research UK London Research Institute, Lincoln's Inn Fields Laboratories, London WC2A 3PX, U.K.
    2. Centre for Cutaneous Research, Institute of Cell and Molecular Science, Barts and the London School of Medicine and Dentistry, Queen Mary, University of London, London E1 2AT, U.K.
    Search for more papers by this author

Yan Gu. Tel.: +44 (0)208 3838528; e-mail: yan.gu@csc.mrc.ac.uk

Summary

Fluorescence resonance energy transfer (FRET) by acceptor photobleaching is a simple but effective tool for measurements of protein–protein interactions. Until recently, it has been restricted to qualitative or relative assessments owing to the spectral bleed-through contamination resulting from fluorescence overlap between the donor and the acceptor. In this paper, we report a quantitative algorithm that combines the spectral unmixing technique with FRET by acceptor photobleaching. By spectrally unmixing the emissions before and after photobleaching, it is possible to resolve the spectral bleed-through and retrieve the FRET efficiency/interaction distance quantitatively. Using a human keratinocyte cell line transfected with cyan fluorescent protein (CFP)- and yellow fluorescent protein (YFP)-tagged Cx26 connexins as an example, FRET information at homotypic gap junctions is measured and compared with well-established methods. Results indicate that the new approach is sensitive, flexible, instrument independent and solely FRET dependent. It can achieve FRET estimations similar to that from a sensitized emission FRET method. This approach has a great advantage in providing the relative concentrations of the donor and the acceptor; this is, for example, very important in the comparative study of cell populations with variable expression levels.

Ancillary