title: A fusion tag enabling optical marking and tracking of proteins and cells by FRET-acceptor photobleaching.
abstract: Combined time-lapse imaging with optical marking of fluorescent proteins (FPs) is a widely used method in studies of the dynamic behaviour of proteins, organelles and cell populations. Most of the approaches have specific limitations as they do not permit simultaneous observation of marked and non-marked molecules, require co-expression of two FP-tagged proteins or rely on oligomerizing FPs. Here we provide a strategy to overcome such limitations with a fluorescence resonance energy transfer-competent tandem fusion tag composed of two FPs. We combine optical marking by acceptor photobleaching with spectral imaging to discriminate between marked and non-marked molecules. Such 'bleach-labelling' may be employed in a broad range of studies for robust real-time tracking of proteins, organelles and cells.
authors.affiliation: Max-Planck-Institut für Immunbiologie, Stübeweg 51, D-79108 Freiburg, Germany.
authors.forename: S
authors.initials: S
authors.lastname: Kuppig
authors.forename: R
authors.initials: R
authors.lastname: Nitschke
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D008856:Microscopy, Fluorescence / Q000379:methods; D055095:Optics and Photonics; D038761:Photobleaching; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D013050:Spectrometry, Fluorescence
chemical_list: D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins