title: Isolation of Escherichia coli release factor-2/ribosome complexes facilitated by efficient crosslinking and sensitive immunodetection of the ligand on the macromolecular particle.
abstract: Complexes between the protein ligand, release factor-2 (RF-2) and the 70S ribosome readily dissociate during purification. To stabilise the weak 70S.RF-2 complex the two components have been crosslinked using dimethyl suberimidate. A sensitive dot blot procedure employing RF-2 specific antibodies and detecting 0.1 ng of factor was developed to improve crosslinking yields. The proportion of the population of ribosomes carrying a release factor was increased several fold to at least 25% as estimated by immunoprecipitation. This combination of a sensitive immunoassay with an efficient crosslinking technique has general application in studies on the binding of a ligand with a macromolecular complex where an antibody against the ligand is available.
authors.affiliation: Department of Biochemistry, University of Otago, Dunedin, New Zealand.
authors.forename: B
authors.initials: B
authors.lastname: Kastner
authors.forename: C N
authors.initials: CN
authors.lastname: Trotman
authors.forename: W P
authors.initials: WP
authors.lastname: Tate
mesh_terms: D002499:Centrifugation, Density Gradient; D004120:Dimethyl Suberimidate / Q000494:pharmacology*; D004926:Escherichia coli / Q000378:metabolism* / Q000648:ultrastructure; D007096:Imidoesters / Q000494:pharmacology*; D008024:Ligands; D010454:Peptide Termination Factors / Q000302:isolation & purification* / Q000378:metabolism; D012270:Ribosomes / Q000187:drug effects / Q000648:ultrastructure*
chemical_list: D007096:Imidoesters; D008024:Ligands; D010454:Peptide Termination Factors; C041460:peptide chain termination release factor 2; D004120:Dimethyl Suberimidate