title: Novel fluorescence labeling and high-throughput assay technologies for in vitro analysis of protein interactions.
abstract: We developed and tested a simple method for fluorescence labeling and interaction analysis of proteins based on a highly efficient in vitro translation system combined with high-throughput technologies such as microarrays and fluorescence cross-correlation spectroscopy (FCCS). By use of puromycin analogs linked to various fluorophores through a deoxycytidylic acid linker, a single fluorophore can be efficiently incorporated into a protein at the carboxyl terminus during in vitro translation. We confirmed that the resulting fluorescently labeled proteins are useful for probing protein-protein and protein-DNA interactions by means of pulldown assay, DNA microarrays, and FCCS in model experiments. These fluorescence assay systems can be easily extended to highly parallel analysis of protein interactions in studies of functional genomics.
authors.affiliation: Department of Applied Chemistry, Faculty of Science and Technology, Keio University, Yokohama 223-8522, Japan.
authors.forename: Nobuhide
authors.initials: N
authors.lastname: Doi
authors.forename: Hideaki
authors.initials: H
authors.lastname: Takashima
authors.forename: Masataka
authors.initials: M
authors.lastname: Kinjo
authors.forename: Kyoko
authors.initials: K
authors.lastname: Sakata
authors.forename: Yuko
authors.initials: Y
authors.lastname: Kawahashi
authors.forename: Yuko
authors.initials: Y
authors.lastname: Oishi
authors.forename: Rieko
authors.initials: R
authors.lastname: Oyama
authors.forename: Etsuko
authors.initials: E
authors.lastname: Miyamoto-Sato
authors.forename: Tatsuya
authors.initials: T
authors.lastname: Sawasaki
authors.forename: Yaeta
authors.initials: Y
authors.lastname: Endo
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Yanagawa
mesh_terms: D004268:DNA-Binding Proteins / Q000032:analysis; D005456:Fluorescent Dyes / Q000378:metabolism*; D007700:Kinetics; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods; D025941:Protein Interaction Mapping / Q000379:methods*; D011691:Puromycin / Q000031:analogs & derivatives / Q000378:metabolism; D013050:Spectrometry, Fluorescence / Q000379:methods; D013194:Staining and Labeling / Q000379:methods*
chemical_list: D004268:DNA-Binding Proteins; D005456:Fluorescent Dyes; D011691:Puromycin