An improved procedure of specific polysome precipitation with antibody.
The specific immunoprecipitation of polysomes prepared from a mouse myeloma, 31C, synthesizing an IgG1 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 mug of the first antibody, 14 mug of antigen added second to the polysomes and 38 mug of the antibody added finally and these were found to precipitate most efficiently one A260 unit of 31C polysomes.
D000818:Animals; D000912:Antibodies, Neoplasm; D000937:Antigen-Antibody Reactions; D000951:Antigens, Neoplasm; D002460:Cell Line; D011232:Chemical Precipitation; D007158:Immunologic Techniques; D051379:Mice; D009194:Myeloma Proteins / Q000276:immunology; D010954:Plasmacytoma; D011092:Polyethylene Glycols / Q000494:pharmacology; D011132:Polyribosomes / Q000276:immunology; D011189:Potassium Chloride / Q000494:pharmacology; D012333:RNA, Messenger / Q000302:isolation & purification*; D012334:RNA, Neoplasm / Q000302:isolation & purification*; D012965:Sodium Chloride / Q000494:pharmacology; D013395:Sucrose / Q000494:pharmacology
D000912:Antibodies, Neoplasm; D000951:Antigens, Neoplasm; D009194:Myeloma Proteins; D012333:RNA, Messenger; D012334:RNA, Neoplasm; D011092:Polyethylene Glycols; D012965:Sodium Chloride; D013395:Sucrose; D011189:Potassium Chloride