2001 Fiscal Year Final Research Report Summary
Sequential analysis of expressed proteins in organelles from rat liver using 1D/2D SDS-PAGE as a clinical molecullar scanner
| Project/Area Number |
12670122
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
General medical chemistry
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| Research Institution | JUNTENDO UNIVERSITY |
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Principal Investigator |
MURAYAMA Kimie Juntendo University, School of Medicine, Associate Professor, 医学部, 助教授 (20053118)
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| Co-Investigator(Kenkyū-buntansha) |
MORITA Masataka Hitachi Koki Co.Ltd., Sales & Promotion Dept. Senior Engineer, 精機事業部, 主任技師
FUJIMURA Tsutomu Juntendo University, School of Medicine, Instructor, 医学部, 助手 (70245778)
SHINDO Noriko Juntendo University, School of Medicine, Instructor, 医学部, 助手 (60095809)
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| Project Period (FY) |
2000 – 2001
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| Keywords | Rat liver proteins / clinical molecular scanner / fractionation of organelles / 1D / 2D SDS-PAGE / Western blotting / peptide mapping / alkylation / アルキレーション |
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| Research Abstract |
In the post-genome era, it is important to understand the function of genomes, which express the proteins in cells, tissues and physiological fluids. The two-dimensional electrophoresis profile (2D SDS-PAGE) of proteins is a. useful tool for separation and characterization ofproteins expressed by genomes. It also might be used for a diagnostic tool such as " marker" proteins for specific disease. Characterization of proteins in tissues is some times difficult to accomplish particularly for proteins in low copy numbers. In the purpose of our study, the expressed protein profiles in organelles of cell or tissues using the ID/ 2D SDS-PAGE were used as a clinical molecular scanner. First we developed a new procedure using freezing-thawing to density gradient solution of Nycodenz for one-step separation of organelles from the rat liver. The density gradient of Nycodenz was prepared from 20% solution in a centrifuge tube by freezing-thawing overnight -20C and at room temperature for a few ho
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urs without the initial centrifugation procedure. Rat liver homogenate, which was removed nuclei and cytosolic fractions, was layered on the Nycodenz gradient solution, centrifuged and separated the subcellular fractions. After fractionation, the protein profile were examined using ID SDS-PAGE. The marker protein of each organelle was confirmed using Western blotting. The expressed proteins in the main fractions of the rat liver were separated on 2D SDS-PAGEs. Reduction and alkylation of cysteinyl residues in proteins is an important process before in gel digestion for proteome analysis. We developed in situ alkylation with acrylamide during SDS gel electrophoresis to yield a thioether derivative, cys-S-beta-propionamide(PAM-cys). The coverage of cysteinyl peptides and total tryptic peptides was increased. And PAM-cys was easy to identify proteins using the databases. The comparison of expressed protein profiles in liver organelles between adult and aging mouse was started by sequential analysis using organelle fractionation, ID/2D SDS-PAGE. Less
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