Systematizing for determination of an amino acid sequence of a micro amount of protein separated by two-dimentional acrylamide gel electrophoresis.
| Project/Area Number |
58870023
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| Research Category |
Grant-in-Aid for Developmental Scientific Research
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| Allocation Type | Single-year Grants |
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| Research Field |
General medical chemistry
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| Research Institution | Niigata University |
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Principal Investigator |
OGATA Kikuo 新潟大学, 医, 教授 (00018285)
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| Project Period (FY) |
1983 – 1985
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| Project Status |
Completed (Fiscal Year 1985)
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| Budget Amount *help |
¥7,100,000 (Direct Cost: ¥7,100,000)
Fiscal Year 1985: ¥1,500,000 (Direct Cost: ¥1,500,000)
Fiscal Year 1984: ¥2,000,000 (Direct Cost: ¥2,000,000)
Fiscal Year 1983: ¥3,600,000 (Direct Cost: ¥3,600,000)
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| Keywords | ribosomal protein / micro sequencer / amino acid composition / cDNA clone / 合成ヌクレオチドプローブ |
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| Research Abstract |
(1) Rat liver ribosomal proteim (S26), separated by two-dimentional polyacrylamide gel electrophoresis was extracted with formic acid, purified by Biogel-P2, was subjected to a microsequencer and the sequence of N-terminal 32 amino acid was determined and used for identification of cDNA clone specific for S26.
(2)Proteins of ribosomal small subunits of Artemia salina separated by two-dimensional acrylamide gel electrophoresis was purified by gel-filtration high performance liquid chromatography (HPLC) and hydrolyzed. Resulting amino acids were dabsylated and subjected to reverse-phase HPLC. The amino acid composition of cytochrome c, ribosomal protein S6 or S8 determined by this method was almost the same as that determined by an amino acid analyzer except for tyrosine. Thus, an amino acid composition of 0.1-1 <micro> g protein is determined by this method and can be used for identification of the cDNA clone specific for the protein.
(3)The cDNA clone for bovine opsin was prepared by screening by colony hybridization with a oligonucleotide probe containing 18-base-long synthetic desoxyribonucleotide.
From these results the systematizing for determination of an amino acid sequence of a micro amount of protein separated by two-dimentional acrylamide gel electrophoresis is performed as follows;
(a)Protein in the gel is extracted with formic acid, purified by Biogel-P2 and subjected to a microsequencer. The partial amino acid sequence thus obtained is used for preparing the probe of oligonucleotides for screening of cDNA clone specific for the protein.
(b)For the identification of a cDNA clone as that specific for the protein, the amino acid sequence determined by the microsequencer described in (a) or the amino acid composition determined by a micro method described in (2) is used.
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Report
(1 results)
Research Products
(8 results)
