J. Biochem, 1984, Vol. 95, No. 2 511-519
© 1984 Japanese Biochemical Society
research-article |
Detection of Calcium Binding Proteins by 45Ca Autoradiography on Nitrocellulose Membrane after Sodium Dodecyl Sulfate Gel Electrophoresis1
Department of Pharmacology, Faculty of Medicine, The University of Tokyo Hongo, Bunkyo-ku, Tokyo 113
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 45Ca 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 µg of calcium binding protein in the starting sample. Under appropriate conditions it was possible to detect only high affinity calcium binding proteins.
1 This work was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan, the Muscular Dystrophy Association of America, and the Tatrochemical Research Foundation.
2 Present address: Laboratory of Neurochemistry, National lnstitute for Physiological Sciences, Myodaiji, Oka-zaki, Aichi 444.
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