The principle of mRNA purification by hybridization to an immobilized DNA fragment was applied to the isolation of mRNA coding for immunoglobulin κ-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 κ-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°C and by elution at 60 °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 κ-chain, was identified as the only translation product.
- diazobenzyloxymethyl- cellulose
- polyacrylamide gel electrophoresis
- RNA-DNA hybridization
- translation in cell- free system
ASJC Scopus subject areas
- Immunology and Allergy