Purification of mRNA for immunoglobulin κ-chains from myeloma and hybridoma cells using hybridization to immobilized complementary DNA

Abstract

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 M r of 25,000, corresponding obviously to the κ-chain, was identified as the only translation product.