title: Immunochemical determination of the cellular content of polypeptide chain release factor RF3 in Escherichia coli.
abstract: Polypeptide chain termination in Escherichia coli is known to require two codon specific release factors, RF1 and RF2. A third factor, RF3, has been described to stimulate the termination. Earlier investigations have estimated the cellular content of factors RF1 and RF2. Two different immunological techniques for measuring the amount of RF3 per cell in crude E coli cell extracts are reported here, using a sensitive immunoblotting method and a sandwich assay by ELISA. Monoclonal murine antibodies and polyclonal rabbit antibodies were raised against extensively purified recombinant E coli RF3. The immunoblotting involves a specific monoclonal antibody (mAb), biotinylated second antibody and finally radioactive iodinated streptavidin. In the sandwich assay polyclonal antibodies are immobilised on a polystyrene surface before addition of crude cell extract; a specific mAb serves as primary antibody and an HRP-labelled anti-mouse Ig as secondary antibody. Both methods are accurate and rapid to perform. The number of RF3 molecules per cell in exponentially growing E coli cells was found to vary considerably according to the K12 strain examined and depended on the culture medium (from 20 to 500 molecules per cell), faster growth being positively correlated with the number of RF3 molecules per cell.
authors.affiliation: Department of Molecular and Structural Biology, Aarhus University, Aarhus C, Denmark.
authors.forename: P
authors.initials: P
authors.lastname: Holst-Hansen
authors.forename: J
authors.initials: J
authors.lastname: Kildsgaard
authors.forename: J
authors.initials: J
authors.lastname: MacDougall
authors.forename: J M
authors.initials: JM
authors.lastname: Palacios Moreno
authors.forename: J
authors.initials: J
authors.lastname: Egebjerg
authors.forename: K K
authors.initials: KK
authors.lastname: Mortensen
authors.forename: H U
authors.initials: HU
authors.lastname: Sperling-Petersen
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000737:chemistry; D001426:Bacterial Proteins / Q000032:analysis* / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000737:chemistry* / Q000254:growth & development; D007106:Immune Sera / Q000096:biosynthesis / Q000737:chemistry; D015151:Immunoblotting; D051379:Mice; D008807:Mice, Inbred BALB C; D010454:Peptide Termination Factors / Q000032:analysis* / Q000276:immunology
chemical_list: D000911:Antibodies, Monoclonal; D001426:Bacterial Proteins; D007106:Immune Sera; D010454:Peptide Termination Factors; C087971:peptide-chain-release factor 3