Isolation of Escherichia coli release factor-2/ribosome complexes facilitated by efficient crosslinking and sensitive immunodetection of the ligand on the macromolecular particle.
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.
Kastner
B
B
Department of Biochemistry, University of Otago, Dunedin, New Zealand.
Trotman
C N
CN
Tate
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WP
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*
D007096:Imidoesters; D008024:Ligands; D010454:Peptide Termination Factors; C041460:peptide chain termination release factor 2; D004120:Dimethyl Suberimidate