Novel fluorescence labeling and high-throughput assay technologies for in vitro analysis of protein interactions.
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.
Doi
Nobuhide
N
Department of Applied Chemistry, Faculty of Science and Technology, Keio University, Yokohama 223-8522, Japan.
Takashima
Hideaki
H
Kinjo
Masataka
M
Sakata
Kyoko
K
Kawahashi
Yuko
Y
Oishi
Yuko
Y
Oyama
Rieko
R
Miyamoto-Sato
Etsuko
E
Sawasaki
Tatsuya
T
Endo
Yaeta
Y
Yanagawa
Hiroshi
H
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*
D004268:DNA-Binding Proteins; D005456:Fluorescent Dyes; D011691:Puromycin