Affinity-purified antibody as a probe of RNA polymerase II subunit structure.
Antibodies reactive against mammalian RNA polymerase II were isolated by affinity chromatography. Serum from a goat immunized with Drosophila RNA polymerase II was passed over a column containing covalently coupled calf thymus RNA polymerase II, and reactive antibodies were eluted at low pH. The affinity-purified antibody was reactive against two large subunits (Mr = 140,000-220,000) and four small subunits (Mr = 16,000-35,000) of RNA polymerase II purified from calf, human, chicken, and Drosophila, as well as two large subunits of wheat germ RNA polymerase II. These findings confirm that RNA polymerase II subunits share conserved antigenic determinants over wide evolutionary distances. The affinity-purified antibody was also used to investigate the presence and structure of RNA polymerase II in human cell extracts capable of carrying out promoter-selective transcription in vitro. The subunit structure of RNA polymerase in the extract appears to be the same as that of purified human RNA polymerase II. In both cases, the human enzyme differs from the calf thymus enzyme in that it contains a large (Mr = 220,000) form of the largest subunit.
D000818:Animals; D000906:Antibodies / Q000302:isolation & purification*; D002417:Cattle; D002642:Chick Embryo; D002846:Chromatography, Affinity; D003429:Cross Reactions; D004330:Drosophila / Q000201:enzymology; D000939:Epitopes / Q000276:immunology; D006041:Goats; D006801:Humans; D008970:Molecular Weight; D011487:Protein Conformation; D012319:RNA Polymerase II / Q000276:immunology*; D013045:Species Specificity
D000906:Antibodies; D000939:Epitopes; D012319:RNA Polymerase II