High-throughput fluorescence labelling of full-length cDNA products based on a reconstituted translation system

Yuko Kawahashi, Nobuhide Doi, Yo Oishi, Chizuru Tsuda, Hideaki Takashima, Tomoya Baba, Hirotada Mori, Takashi Ito, Hiroshi Yanagawa

Research output: Contribution to journalArticle

3 Citations (Scopus)

Abstract

Although recent advances in fluorescence-based technologies, such as protein microarrays, have made it possible to analyse more than 10,000 proteins at once, there is a bottleneck in the step of preparation of large numbers of fluorescently labelled proteins for the comprehensive analysis of protein-protein interactions. Here we describe two independent methods for high-throughput fluorescence-labelling of full-length cDNA products at their C-termini using a reconstituted translation system containing fluorescent puromycin. For the first method, release factor-free systems were used. For the second method, stop codons were excluded from cDNAs by using a common mismatch primer in mutagenic PCR. These methods yielded large numbers of labelled proteins from cDNA sets of various organisms, such as mouse, yeast and Escherichia coli.

Original languageEnglish
Pages (from-to)19-24
Number of pages6
JournalJournal of Biochemistry
Volume141
Issue number1
DOIs
Publication statusPublished - 2007 Jan

Fingerprint

Labeling
Complementary DNA
Fluorescence
Throughput
Proteins
Puromycin
Protein Array Analysis
Terminator Codon
Microarrays
Yeast
Escherichia coli
Yeasts
Technology
Polymerase Chain Reaction

Keywords

  • Cell-free protein synthesis
  • Fluorescence labelling
  • Proteomics
  • Puromycin
  • Release factor

ASJC Scopus subject areas

  • Biochemistry

Cite this

High-throughput fluorescence labelling of full-length cDNA products based on a reconstituted translation system. / Kawahashi, Yuko; Doi, Nobuhide; Oishi, Yo; Tsuda, Chizuru; Takashima, Hideaki; Baba, Tomoya; Mori, Hirotada; Ito, Takashi; Yanagawa, Hiroshi.

In: Journal of Biochemistry, Vol. 141, No. 1, 01.2007, p. 19-24.

Research output: Contribution to journalArticle

Kawahashi, Y, Doi, N, Oishi, Y, Tsuda, C, Takashima, H, Baba, T, Mori, H, Ito, T & Yanagawa, H 2007, 'High-throughput fluorescence labelling of full-length cDNA products based on a reconstituted translation system', Journal of Biochemistry, vol. 141, no. 1, pp. 19-24. https://doi.org/10.1093/jb/mvm003
Kawahashi, Yuko ; Doi, Nobuhide ; Oishi, Yo ; Tsuda, Chizuru ; Takashima, Hideaki ; Baba, Tomoya ; Mori, Hirotada ; Ito, Takashi ; Yanagawa, Hiroshi. / High-throughput fluorescence labelling of full-length cDNA products based on a reconstituted translation system. In: Journal of Biochemistry. 2007 ; Vol. 141, No. 1. pp. 19-24.
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