2002 Fiscal Year Final Research Report Summary
Establishment of protein analysis methods coupled with laser-microdissection to identify} subnanogram amounts of proteins in tissue microstructures.
| Project/Area Number |
12470146
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Neurology
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| Research Institution | National Institute of Neuroscience, National Center of Neurology and Psychiatry |
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Principal Investigator |
KANEKO Kiyotoshi National Institute of Neuroscience, Director, Division of Cortical Function Disorders, 神経研究所・疾病研究第七部, 部長 (10251513)
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| Project Period (FY) |
2000 – 2002
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| Keywords | laser microdissection / scFv (single chain variable fragment) / tissue section / in situ phage screening / nanogram antigen / in situ western blot |
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| Research Abstract |
We have developed three protein analysis methods coupled with laser-microdissection to identify subnanogram amounts of proteins in tissue microstructures. ( 1 ) In situ Phage Screening (ISPS). By coupling the laser-microdissection with phage display screening system, we obtained several single chain antibody variable region fragments (scFv) against human muscle proteins (major components: actin, myosin and tropomyosin α; a minor component, α-actinin 2) on microdissected frozen tissue thin sections (5,000 μm^2). The antigens were determined by immunoscreening of muscle-specific expression library, 2D western blot and MALDI-TOF-MS. (2) In situ Mass Spectrometry (ISMS). By MALDI-TOF-MS analysis of microdissected tissue microstructures, we successfully obtained fingerprinting using the same amount of tissue samples used in ISPS. (3) In situ Western Blotting (ISWB). Subnanogram amount of proteins from microdissected tissue sections were available for western blot analysis. We microdissected Pick bodies, deposits accumulated perinuclearly in neurons of patients with Pick's disease, and conducted high sensitive western blot analysis with anti-phosphorylated tau antibodies. Consequently, we observed novel Pick body-specific abnormally phosphorylated tau molecules. Tissue-specifically accumulated intracellular or extracellular deposits might be involved in the causes of various diseases. We have continued to determine the protein components in disease-specific deposits by our microdissection-coupled protein analysis methods.
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