Fluorescence Resonance Energy Transfer-Based Assay for DNA-Binding Protein Tagged by Green Fluorescent Protein
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概要
- 論文の詳細を見る
Specific interaction between green fluorescent protein (GFP)-tagged human α- or γ-enolase97-242 (α or γENO97-242) and the rhodamine-labeled DNA fragment containing the c-myc P2 promoter was detected by a fluorescence resonance energy transfer (FRET)-based assay, designated as a “real-time FRET assay.” The approach of donor (GFP) and acceptor (rhodamine) was caused by the association between ENO97-242 and the c-myc P2 promoter, and the time-dependent increase in fluorescence intensity of the reaction mixture was observed at ex=400 nm and em=590 nm. The relative affinity (Ras) of ENO97-242 mutants to the wild type was investigated with a real-time FRET assay, and it was clarified that the amino acids that participated in the interaction existed comparatively broadly. Although it was difficult to measure the absolute value of the affinity for the binding protein by using this method, it was possible to investigate the relative affinity of mutants for the wild type. A real-time FRET assay using the GFP-tagged protein could be used as not only a qualitative, but also as a quantitative analysis, this being the best for investigating the key amino acids in binding proteins.
- 社団法人 日本農芸化学会の論文
- 2006-08-23
著者
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Tsuchida Shirou
Department of Molecular Biosciences, School of Pharmaceutical Sciences, Health Sciences University o
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AOKI Takashi
Department of Molecular Biosciences, School of Pharmaceutical Sciences, Health Sciences University o
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Ozaki Hiroyuki
Department of Biotechnology, Graduate School of Engineering, Osaka University
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WATABE Hiroyuki
Department of Internal Medicine, Kitasato University School of Medicine
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Tsuchida Shirou
Department Of Biochemistry Faculty Of Pharmaceutical Sciences Health Sciences University Of Hokkaido
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Ozaki Hiroyuki
Department Of Biochemistry Faculty Of Pharmaceutical Sciences Health Sciences University Of Hokkaido
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IMAMURA Tomoko
Department of Biochemistry, Faculty of Pharmaceutical Sciences, Health Sciences University of Hokkai
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IDEUCHI Hideki
Department of Biochemistry, Faculty of Pharmaceutical Sciences, Health Sciences University of Hokkai
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Ideuchi Hideki
Department Of Biochemistry Faculty Of Pharmaceutical Sciences Health Sciences University Of Hokkaido
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Imamura Tomoko
Department Of Biochemistry Faculty Of Pharmaceutical Sciences Health Sciences University Of Hokkaido
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Watabe Hiroyuki
Department Of Biochemistry Faculty Of Pharmaceutical Sciences Health Sciences University Of Hokkaido
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Aoki Takashi
Department Of Biochemistry Faculty Of Pharmaceutical Sciences Health Sciences University Of Hokkaido
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Aoki Takashi
Department Of Aquatic Biosciences Tokyo University Of Fisheries
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Aoki Takashi
Department Of Aquatic Bioscience Tokyo University Of Fisheries
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Watabe Hiroyuki
Department of Applied Chemistry, Faculty of Engineering, Yamagata University
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