title: Development of a Split SNAP-CLIP Double Labeling System for Tracking Proteins Following Dissociation from Protein-Protein Complexes in Living Cells.
abstract: The split SNAP-tag protein-fragment complementation assay (PCA) is a useful tool for imaging protein-protein interactions (PPIs) in living cells. In contrast to conventional methods employed for imaging PPIs, the split SNAP-tag PCA enables tracking of proteins following dissociation from protein-protein complexes. A limitation of this system, however, is that it only allows for labeling and tracking of one of the proteins forming the protein-protein complex. To track both proteins forming a protein-protein complex, each protein needs to be appropriately labeled. In this study, a split SNAP-CLIP double labeling system is developed and applied for tracking of each protein forming a protein-protein complex. As a proof-of concept, FM protein for PPIs and protein kinase C alpha (PKCα) for translocation are introduced to a split SNAP-CLIP double labeling system. The results show a split SNAP-CLIP double labeling system enables labeling of both proteins in a protein-protein complex and subsequent tracking of each of the proteins following dissociation from the protein-protein complexes in living cells.
authors.affiliation: Department of Life Science and Technology, School of Life Science and Technology, Tokyo Institute of Technology . 4259 Nagatsuta, Midori-ku, Yokohama 226-8502, Japan.
authors.forename: Masayasu
authors.initials: M
authors.lastname: Mie
authors.affiliation: Department of Life Science and Technology, School of Life Science and Technology, Tokyo Institute of Technology . 4259 Nagatsuta, Midori-ku, Yokohama 226-8502, Japan.
authors.forename: Tatsuhiko
authors.initials: T
authors.lastname: Naoki
authors.affiliation: Department of Life Science and Technology, School of Life Science and Technology, Tokyo Institute of Technology . 4259 Nagatsuta, Midori-ku, Yokohama 226-8502, Japan.
authors.forename: Eiry
authors.initials: E
authors.lastname: Kobatake
mesh_terms: D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes / Q000737:chemistry; D006367:HeLa Cells; D006801:Humans; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D010957:Plasmids / Q000235:genetics / Q000378:metabolism*; D054730:Protein Interaction Domains and Motifs; D051571:Protein Kinase C-alpha / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry; D022061:Tacrolimus Binding Protein 1A / Q000235:genetics / Q000378:metabolism
chemical_list: D005456:Fluorescent Dyes; D008562:Membrane Glycoproteins; D011993:Recombinant Fusion Proteins; D051571:Protein Kinase C-alpha; D022061:Tacrolimus Binding Protein 1A