Generation of monoclonal antibodies to RNA polymerase II for the identification of transcriptional factors.
Monoclonal antibodies were raised against a partially purified RNA polymerase II preparation from hen oviduct. Hybridomas were screened using enzyme-linked immunosorbent assays, and those producing antibodies against the RNA polymerase II preparation were cloned twice. The specificity of monoclonal antibodies for RNA polymerase II was determined by three types of assays: first, by immunoblot assay; second, by removal of enzyme activity using indirect immunoprecipitation; and third, by affinity column chromatography. Several monoclonal antibodies were identified. One of them, 4C3-21 was characterized in detail here. This monoclonal antibody recognized the native form of RNA polymerase II from chicken oviduct, calf thymus, and HeLa cells, but it did not recognize Escherichia coli RNA polymerase. In addition, after interacting with RNA polymerase, this antibody did not inhibit total RNA synthesis nor specific initiation of transcription on cloned ovalbumin fragments in vitro. Finally, since the antibody did not react with polymerase subunits separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, we deduced that the antibody interacted with the native form of the enzyme at a site which is not involved in initiation or elongation of RNA synthesis. This allowed us to undertake an unique approach utilizing antibody affinity column chromatography for purification of transcriptional factors. Using this approach, we demonstrated that certain transcription factor(s) were associated with RNA polymerase II and could be absorbed to an affinity column as an RNA polymerase-transcriptional factor complex. Differential elution can be carried out subsequently. Therefore, this antibody column should prove to be very useful for the purification of transcriptional regulatory factor(s) which bind to RNA polymerase.
D000818:Animals; D000911:Antibodies, Monoclonal; D002645:Chickens; D003429:Cross Reactions; D004054:Diethylstilbestrol / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000201:enzymology; D005260:Female; D051379:Mice; D008807:Mice, Inbred BALB C; D008970:Molecular Weight; D010047:Ovalbumin / Q000235:genetics; D010057:Oviducts / Q000201:enzymology; D012319:RNA Polymerase II / Q000276:immunology*; D014157:Transcription Factors / Q000032:analysis*; D014158:Transcription, Genetic
D000911:Antibodies, Monoclonal; D014157:Transcription Factors; D004054:Diethylstilbestrol; D010047:Ovalbumin; D012319:RNA Polymerase II