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An Improved Procedure of Specific Polysome Precipitation with Antibody
Author(s) -
Ono Masao,
Kawakami Masaya
Publication year - 1976
Publication title -
japanese journal of microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.664
H-Index - 70
eISSN - 1348-0421
pISSN - 0021-5139
DOI - 10.1111/j.1348-0421.1976.tb00976.x
Subject(s) - polysome , immunoprecipitation , antibody , chemistry , antigen , ribosome , biochemistry , microbiology and biotechnology , biology , immunology , rna , gene
The specific immunoprecipitation of polysomes prepared from a mouse myeloma, 31C, synthesizing an IgG 1 immunoglobulin has been investigated. A reported method in which polysomes were coprecipitated by sequential addition of antibody to 31C myeloma protein, antigen (i.e., the 31C protein) and again the antibody, was used. Salt concentration greatly affected the immunoprecipitation of polysomes. In the presence of 100 mm KCl or NaCl, 10–20% of myeloma polysomes and only 1% of mouse liver polysomes were precipitated with the antibody to myeloma protein. On the other hand, 90% of the both polysomes were precipitated by the same antibody at a salt concentration of 10 mm. Triton X‐100 and sucrose had little effect on preventing nonspecific binding of immunoglobulin to ribosomes. Experiments were carried out to obtain an optimal ratio of the amount of polysome to that of antibody and antigen to be added for the coprecipitation of polysomes. To date we have tried 25 μg of the first antibody, 14 μg of antigen added second to the polysomes and 38 μg of the antibody added finally and these were found to precipitate most efficiently one A 260 unit of 31C polysomes.