Novel Fluorescence Labeling and High-Throughput Assay Technologies for In Vitro Analysis of Protein Interactions
- Nobuhide Doi1,
- Hideaki Takashima1,2,
- Masataka Kinjo3,
- Kyoko Sakata2,
- Yuko Kawahashi1,4,
- Yuko Oishi1,
- Rieko Oyama1,
- Etsuko Miyamoto-Sato1,
- Tatsuya Sawasaki5,
- Yaeta Endo5, and
- Hiroshi Yanagawa1,6
- 1Department of Applied Chemistry, Faculty of Science and Technology, Keio University, Yokohama 223-8522, Japan; 2Mitsubishi Kagaku Institute of Life Sciences, Machida 194-8511, Japan; 3Research Institute for Electronic Science, Hokkaido University, Sapporo 060-0812, Japan; 4Tsukuba Technical Center, Ikeda Scientific Corporation, Tsukuba 305-0062, Japan; 5 Department of Applied Chemistry, Faculty of Engineering, Ehime University, Matsuyama 790-8577, Japan
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.
[Online supplementary material available at http://www.genome.org.]
Footnotes
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↵6 Corresponding author.
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E-MAIL hyana{at}applc.keio.ac.jp.; FAX 81-45-566-1440.
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Article and publication are at http://www.genome.org/cgi/doi/10.1101/gr.218802.
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- Received October 11, 2001.
- Accepted December 14, 2001.
- Cold Spring Harbor Laboratory Press
