| Project/Area Number |
16310146
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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 |
Living organism molecular science
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| Research Institution | University of Tokyo |
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Principal Investigator |
OHMI Shinobu The University of Tokyo, Institute of Medical Science, Associate Professor, 医科学研究所, 助教授 (20160046)
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| Project Period (FY) |
2004 – 2006
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| Project Status |
Completed (Fiscal Year 2006)
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| Budget Amount *help |
¥13,600,000 (Direct Cost: ¥13,600,000)
Fiscal Year 2006: ¥4,200,000 (Direct Cost: ¥4,200,000)
Fiscal Year 2005: ¥4,200,000 (Direct Cost: ¥4,200,000)
Fiscal Year 2004: ¥5,200,000 (Direct Cost: ¥5,200,000)
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| Keywords | protein / carboxyl terminus / antibody / proteome / amino acid residue / protease / proteolysis / アミノ酸残基 / 構造解析 |
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| Research Abstract |
Cleavage-site directed antibodies, specific to new termini of a polypeptide generated by limited proteolysis, have been used for in situ analysis of proteolytic reaction. Such antibodies can be obtained by immunizing animals with terminus-mimicking synthetic peptide as an immunogenic hapten. Recently, novel substrates for caspases were successfully discovered in apoptotic cells using cleavage-site directed antibodies. We have been investigating antigen-binding specificity for these antibodies by means of peptide libraries. Polyclonal cleavage-site directed antibodies raised in rabbits showed fuzzy binding profiles for second to the C-terminal residue, although the antibodies strictly bound a peptide when Asp was located in its C terminus. However, the antibodies were less specific to the internal region farther away from the C terminus. Thus, we prepared monoclonal antibodies for the C-terminal region of a caspase-catalyzed polypeptide, and re-examined their antigen-binding properties. Some monoclonal antibodies were specific for C-terminal two residues, suggesting that a set of hundreds of such antibodies may be used for C-terminal analysis of a protein. For example, immunoblot analysis of electorophoretically separated proteins enable us to categorize them by C-terminal two residues. We can show in this study cleavage-site directed antibodies are possibly utilized for a proteomic tool in the future generation.
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