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- Title
High-throughput Fluorescence Labelling of Full-length cDNA Products Based on a Reconstituted Translation System.
- Authors
Kawahashi, Yuko; Doi, Nobuhide; Oishi, Yo; Tsuda, Chizuru; Takashima, Hideaki; Baba, Tomoya; Mori, Hirotada; Ito, Takashi; Yanagawa, Hiroshi
- 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.
- Subjects
FLUORESCENCE; PROTEIN microarrays; ESCHERICHIA coli; DNA; BIOCHEMISTRY
- Publication
Journal of Biochemistry, 2007, Vol 141, Issue 1, p19
- ISSN
0021-924X
- Publication type
Article
- DOI
10.1093/jb/mvm003