In vitro protein microarrays for detecting protein-protein interactions: Application of a new method for fluorescence labeling of proteins

Yuko Kawahashi, Nobuhide Doi, Hideaki Takashima, Chizuru Tsuda, Yuko Oishi, Rieko Oyama, Masato Yonezawa, Etsuko Miyamoto-Sato, Hiroshi Yanagawa

Research output: Contribution to journalArticle

65 Citations (Scopus)

Abstract

Protein microarrays or proteome chips are potentially powerful tools for comprehensive analysis of protein-protein interactions. In interaction analysis, a set of immobilized proteins is arrayed on slides and each slide is probed with a set of fluorescently labeled proteins. Here we have developed and tested an in vitro protein microarray, in which both arraying and probing proteins were prepared by cell-free translation. The in vitro synthesis of fluorescently labeled proteins was accomplished by a new method: a fluorophore-puromycin conjugate was incorporated into a protein at the C-terminus on the ribosome. The resulting fluorescently labeled proteins were confirmed to be useful for probing protein-protein interactions on protein microarrays in model experiments. Since the in vitro protein microarrays can easily be extended to a high-throughput format and also combined with in vitro display technologies such as the streptavidin-biotin linkage in emulsions method (Doi and Yanagawa, FEBS Lett. 1999, 457, 227-230), our method should be useful for large-scale analysis of protein-protein interactions.

Original languageEnglish
Pages (from-to)1236-1243
Number of pages8
JournalProteomics
Volume3
Issue number7
DOIs
Publication statusPublished - 2003 Jul 1

Keywords

  • Cell-free protein synthesis
  • Fluorescence labeling
  • Protein microarrays
  • Protein-protein interactions
  • Puromycin technology

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology

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  • Cite this

    Kawahashi, Y., Doi, N., Takashima, H., Tsuda, C., Oishi, Y., Oyama, R., Yonezawa, M., Miyamoto-Sato, E., & Yanagawa, H. (2003). In vitro protein microarrays for detecting protein-protein interactions: Application of a new method for fluorescence labeling of proteins. Proteomics, 3(7), 1236-1243. https://doi.org/10.1002/pmic.200300444