Characterization of U small nuclear RNA-associated proteins.
Differential immunoaffinity chromatography using a combination of autoimmune antibodies allows for the rapid bulk separation of specific small nuclear ribonucleoproteins (snRNPs). Passage of a HeLa cell extract over a column constructed of human anti-Sm autoantibodies results directly in the elution of complexes containing the small nuclear RNA species, U1, U2, U4, U5, and U6, and nine major polypeptides of molecular weight 69,000, 32,000, 27,000, 26,000, 18,500, 13,000, 11,000 doublet, and less than 10,000. Passage of crude extracts through a column bearing murine monoclonal antibodies directed against the 69,000 molecular weight (U1)RNP peptide gives an enriched population of U1 snRNP particles in the retained material. When the flowthrough material from the (U1)RNP column is passed through an anti-Sm column, the retained material is enriched in U2, U4, U5 plus U6 snRNP complex. The 69,000, 32,000, and 18,500 molecular weight polypeptides are confined to the U1 fraction while the remaining proteins are recovered in both fractions. The procedure is simple and rapid, producing complexes with a high degree of resolution and in sufficient yield to provide a ready source of snRNP complexes for functional studies.
D000911:Antibodies, Monoclonal; D002846:Chromatography, Affinity / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel; D006367:HeLa Cells / Q000032:analysis; D006801:Humans; D008970:Molecular Weight; D010761:Phosphorus Radioisotopes; D012313:RNA / Q000302:isolation & purification; D012342:RNA, Small Nuclear; D012261:Ribonucleoproteins / Q000096:biosynthesis / Q000302:isolation & purification*; D017411:Ribonucleoproteins, Small Nuclear
D000911:Antibodies, Monoclonal; D010761:Phosphorus Radioisotopes; D012342:RNA, Small Nuclear; D012261:Ribonucleoproteins; D017411:Ribonucleoproteins, Small Nuclear; D012313:RNA