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Published online before print February 15, 2002, 10.1101/gr.218802
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Vol. 12, Issue 3, 487-492, March 2002

METHODS
Novel Fluorescence Labeling and High-Throughput Assay Technologies for In Vitro Analysis of Protein Interactions

Nobuhide Doi,1 Hideaki Takashima,1,2 Masataka Kinjo,3 Kyoko Sakata,2 Yuko Kawahashi,1,4 Yuko Oishi,1 Rieko Oyama,1 Etsuko Miyamoto-Sato,1 Tatsuya Sawasaki,5 Yaeta Endo,5 and Hiroshi Yanagawa1,6

1 Department of Applied Chemistry, Faculty of Science and Technology, Keio University, Yokohama 223-8522, Japan; 2 Mitsubishi Kagaku Institute of Life Sciences, Machida 194-8511, Japan; 3 Research Institute for Electronic Science, Hokkaido University, Sapporo 060-0812, Japan; 4 Tsukuba Technical Center, Ikeda Scientific Corporation, Tsukuba 305-0062, Japan; 5  Department of Applied Chemistry, Faculty of Engineering, Ehime University, Matsuyama 790-8577, Japan

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.]

6 Corresponding author.

12:487-492 ©2002 by Cold Spring Harbor Laboratory Press  ISSN 1088-9051/02 $5.00
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