Purification of mouse immunoglobulin heavy-chain messenger RNAs from total myeloma tumor RNA

Eur J Biochem. 1980 Jun;107(2):303-14. doi: 10.1111/j.1432-1033.1980.tb06030.x.


A procedure is described for the large-scale purification of light (L) and heavy (H) chain mRNAs from plasmacytomas produced in mice. Intact RNA is selectively precipitated in high yield from frozen tumors homogenized in 3 M LiCl and 6 M urea. L and H-chain mRNAs were purified by oligo(dT)-cellulose chromatography and either sucrose gradient centrifugation in conditions preventing aggregation or by means of high-resolution preparative gel electrophoresis under non-denaturing conditions. gamma 2a and alpha H-chain mRNAs sedimented as major components at 15.5 S and 16.5 S respectively, when L-chain mRNAs sedimented as 12-S species. H-chain mRNAs isolated by continuous elution during preparative gel electrophoresis were completely separated from both L-chain mRNA and residual 18-S rRNA, and migrated as single components of 1900 +/- 50 nucleotides on analytical denaturing gels. The partially purified H-chain mRNAs were translated into major components of molecular weights of 56,000 (gamma 2a) and 60,000 (alpha) in an mRNA-dependent rabbit reticulocyte lysate, whereas L-chain mRNAs yielded polypeptides of molecular weights of 25,000 (gamma) and 27,000 (chi). Up to 95% of the translation products directed by the purified mRNAs were immunoprecipitated using specific antisera. The purity of L and H-chain mRNAs was assessed by hybridization of corresponding cDNAs with excess recombinant plasmid DNA. The results indicated a minimum purity of 47% (gamma 2a), 62% (alpha), for H-chain mRNAs and 60% (chi), for L-chain mRNAs.

MeSH terms

  • Animals
  • Centrifugation, Density Gradient / methods
  • Electrophoresis, Disc / methods
  • Immunoglobulin Heavy Chains / biosynthesis*
  • Mice
  • Plasmacytoma / analysis
  • Poly A / metabolism
  • Protein Biosynthesis
  • RNA, Messenger / isolation & purification*


  • Immunoglobulin Heavy Chains
  • RNA, Messenger
  • Poly A