Novel fluorescence labeling and high-throughput assay technologies for in vitro analysis of protein interactions

Nobuhide Doi, Hideaki Takashima, Masataka Kinjo, Kyoko Sakata, Yuko Kawahashi, Yuko Oishi, Rieko Oyama, Etsuko Miyamoto-Sato, Tatsuya Sawasaki, Yaeta Endo, Hiroshi Yanagawa

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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.

Original languageEnglish
Pages (from-to)487-492
Number of pages6
JournalGenome Research
Volume12
Issue number3
DOIs
Publication statusPublished - 2002 Mar 27

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ASJC Scopus subject areas

  • Genetics
  • Genetics(clinical)

Cite this

Doi, N., Takashima, H., Kinjo, M., Sakata, K., Kawahashi, Y., Oishi, Y., Oyama, R., Miyamoto-Sato, E., Sawasaki, T., Endo, Y., & Yanagawa, H. (2002). Novel fluorescence labeling and high-throughput assay technologies for in vitro analysis of protein interactions. Genome Research, 12(3), 487-492. https://doi.org/10.1101/gr.218802. Article published online before print in February 2002