Purification of RNA using an anti-RNA monoclonal antibody.
Studies of the synthesis and modification of RNA employ many types of in vitro reactions. Often, the RNA product must be concentrated or purified away from other reaction components such as salts, unincorporated nucleotides, protein, or DNA. Here I describe an immunological approach suitable for the isolation of RNA from in vitro reactions. A variety of RNAs of differing size and nucleotide sequence were immunoprecipitated with a monoclonal antibody specific for RNA. RNA binding took place in seconds with nearly quantitative recoveries. Immunoprecipitation was more efficient than ethanol precipitation in removing unincorporated nucleotides. Proteins which do not bind to RNA remained soluble. The immunoprecipitated RNA sample was solubilized directly with a buffered solution suitable for gel electrophoresis under denaturing conditions. Thus, RNAs can be rapidly concentrated for electrophoresis in a single step. Antibody-RNA binding was reversible under nondenaturing conditions in the presence of excess rRNA. This procedure serves as a novel means of purifying RNA and RNA-binding proteins from in vitro reactions.
Reines
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Department of Biochemistry, Emory University School of Medicine, Atlanta, Georgia 30322.
D000818:Animals; D000911:Antibodies, Monoclonal; D004586:Electrophoresis / Q000379:methods; D000431:Ethanol; D007150:Immunohistochemistry / Q000379:methods; D010761:Phosphorus Radioisotopes; D012313:RNA / Q000276:immunology / Q000302:isolation & purification*; D051381:Rats; D012995:Solubility; D014158:Transcription, Genetic / Q000502:physiology
D000911:Antibodies, Monoclonal; D010761:Phosphorus Radioisotopes; D000431:Ethanol; D012313:RNA