Purification of mRNA for immunoglobulin kappa-chains from myeloma and hybridoma cells using hybridization to immobilized complementary DNA.
The principle of mRNA purification by hybridization to an immobilized DNA fragment was applied to the isolation of mRNA coding for immunoglobulin kappa-chains of mouse myeloma MOPC 21 and mouse hybridoma PTF-02. The DNA fragment comprising the 3'-untranslated region and a part of the constant region of the kappa-chain gene was covalently attached to diazobenzyloxymethyl-cellulose and used as an affinity adsorbent. A homogeneous 14S mRNA species was obtained by hybridization of total mRNA to the affinity adsorbent at 52 degrees C and by elution at 60 degrees C. Addition of the purified mRNA to a fractionated cell-free translation system resulted in a significant increase in the radioactivity immunoprecipitated by pig anti-mouse immunoglobulin antibodies. A single radioactive polypeptide of apparent Mr of 25,000, corresponding obviously to the kappa-chain, was identified as the only translation product.
D000818:Animals; D004247:DNA / Q000235:genetics; D006825:Hybridomas / Q000276:immunology*; D007147:Immunoglobulin Light Chains / Q000235:genetics*; D007145:Immunoglobulin kappa-Chains / Q000235:genetics*; D051379:Mice; D009101:Multiple Myeloma / Q000235:genetics / Q000276:immunology*; D009693:Nucleic Acid Hybridization; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000235:genetics / Q000302:isolation & purification*; D012334:RNA, Neoplasm / Q000302:isolation & purification
D007147:Immunoglobulin Light Chains; D007145:Immunoglobulin kappa-Chains; D012333:RNA, Messenger; D012334:RNA, Neoplasm; D004247:DNA