1985 Fiscal Year Final Research Report Summary
Detection of Calcium Binding Proteins on Nitrocellulose Membrane
| Project/Area Number |
57880034
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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 |
分子遺伝学・分子生理学
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| Research Institution | The University of Tokyo
Okazaki National Research Institutes |
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Principal Investigator |
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| Project Period (FY) |
1982 – 1985
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| Keywords | Calcium Binding Protein / アフィニティ分析 / ウエスタンブロット |
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| Research Abstract |
A new simple method of detecting calcium binding proteins in a protein mixture is described. A sample which might include calcium binding proteins was subjected to SDS-polyacrylamide gel electrophoresis and then electrophoretically transferred to a nitrocellulose membrane. The membrane was then incubated with <^(45)Ca> to detect calcium binding proteins as radioactive bands by autoradiography.
Purified troponin-C, calmodulin, myosin DTNB light chain, and parvalbumin were clearly identified by this method. In the whole homogenate of chicken skeletal muscle, myosin DTNB light chain, troponin-C, and 55K calcium binding protein were found to be radioactive. In the frog skeletal muscle, small molecular weight proteins of approximately 13-15K and 70K protein appeared to be the calcium binding proteins. In the case of the carp skeletal muscle, small molecular weight proteins including parvalbumin and two proteins of about 80K seemed to bind calcium ion. Two high molecular weight calcium binding proteins were present in the scallop striated muscle.
The procedure described can be completed within 24 h and can detect as little as 2 <micro> g of calcium binding protein in the starting sample. Under appropriate conditions it was possible to detect only high affinity calcium binding proteins.
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