Protein carbonyl detection by two-dimensional fluorescence difference gel electrophoresis
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概要
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Oxidative stress is implicated in a broad variety of chronic and acute diseases, including Alzheimers disease, arteriosclerosis and diabetes. In order to study oxidative stress at the proteome level proteins were labeled with Cy2NHS (the N-hydroxyl succinimidyl ester derivative of a cyanine dye reactive with the ε-amino group of lysine residues), or with Cy3- and Cy5HZ (hydrazide derivatives of the dyes reactive with protein carbonyls) and analyzed by two-dimensional fluorescence difference gel electrophoresis (2D-DIGE). Two samples were used for testing the CyHZ labeling; one is low molecular mass marker proteins artificially oxidized by NaOCl and the other renal cortex extracts of 30-week-old diabetes model OLETF (Otsuka Long-Evans Tokushima Fatty) and control LETO (Long-Evans Tokushima Otsuka) rats. Spot volumes Vni and contrasts cni of 2D-DIGE images were quantified following background subtraction, where the suffices n (=2, 3 or 5) and i (=1, 2, ...) represent the Cy-signal and the Cy2-detected spot identification numbers, respectively. The contrasts c3i and c 5i were on average about 0.53 times as high as c2i. Taking the contrast into account, a simple parameter qni was proposed for judging the quality of spot images. The difference in-gel analysis (DIA) algorithm should be used in combination with the quality parameters to identify statistical outliers as candidates for abnormally carbonylated proteins. Inspection of the background flatness around the candidate spots is also required before making a conjecture as to whether the candidate protein is abnormally oxidized.
- 2008-03-01
著者
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OH-ISHI Masamichi
Department of Physics, School of Science
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MAEDA Tadakazu
Department of Physics, School of Science
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SAWADA Akira
Department of Ophthalmology, Gifu University School of Medicine
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Oh-ishi Masamichi
Department Of Physics Kitasato University School Of Science
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Maeda Tadakazu
Laboratory Of Biomolecular Dynamics Department Of Physics Kitasato University School Of Science
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Maeda Tadakazu
Graduate School Of Science Kitasato University
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Maeda Tadakazu
Department Of Physics Faculty Of Science Nagoya University:mitsubishi-kasei Institute Of Life Scienc
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UENO Takeshi
Department of Molecular Medicine, School of Medicine, Kitasato University
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KODERA Yoshio
Departments of Physics, Kitasato University School of Science
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Kodera Yoshio
Laboratory Of Biomolecular Dynamics Department Of Physics Kitasato University School Of Science
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KAWASHIMA YUSUKE
Department of Surgery, Kurume University School of Medicine
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Kawashima Yusuke
Laboratory Of Biomolecular Dynamics Department Of Physics Kitasato University School Of Science
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Kawashima Yusuke
Department Of Surgery Kurume University School Of Medicine
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Matsui Takashi
Department Of Physics School Of Science Kitasato University
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HARUTA SATOH
Department of Molecular Diagnosis, Graduate School of Medicine, Chiba University
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Haruta Satoh
Department Of Molecular Diagnosis Graduate School Of Medicine Chiba University
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Sawada Akira
Department Of Ophthalmology Gifu University Graduate School Of Medicine
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Kodera Yoshio
Department Of Physics Kitasato University School Of Science
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Maeda Tadakazu
Department Of Physics Kitasato University School Of Science
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Kawashima Yusuke
Department Of Physics Kitasato University School Of Science
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Sawada Akira
Department Of Physics Kitasato University School Of Science
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Sawada Akira
Department Of Applied Materials Science Muroran Institute Of Technology
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Ueno Takeshi
Department Of Pharmacology And Toxicology Kyorin University School Of Medicine
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Ueno Takeshi
Department Of Electrical And Electronics Engineering Chuo University
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