A rapid immunoprecipitation assay for neomycin phosphotransferase II expression in transformed bacteria and plant tissues.
Anti-kanamycin antibodies produced in rabbits, following coupling of the antibiotic to bovine serum albumin, were used to immunoprecipitate radioactively labelled phosphorylated kanamycin from transformed bacterial or plant extracts in a novel assay system, for the detection of neomycin phosphotransferase II (NPTII) activity. Radioactive counts in the immunoprecipitated pellet give a semiquantitative measure of the kanamycin phosphorylation and hence the amount of NPTII activity. This assay is sensitive, uses very small amounts of radioactivity, and is very rapid, allowing many samples to be processed within a few hours. Immunoprecipitated counts from reactions with bacteria carrying a kanamycin resistance gene or from tobacco and Brassica napus plants transformed with NPTII gene-containing vectors were consistently higher than counts from nontransformed controls. Results obtained with this assay correlate well with those from the previously described gel overlay and dot-blot assays, but can be obtained in an appreciably shorter time frame.
Baszczynski
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Allelix Crop Technologies Inc., Mississauga, Ont., Canada.
D000818:Animals; D000918:Antibody Specificity; D001937:Brassica / Q000032:analysis; D003432:Cross-Linking Reagents; D004926:Escherichia coli / Q000032:analysis; D007106:Immune Sera / Q000096:biosynthesis; D007118:Immunoassay; D007612:Kanamycin / Q000276:immunology; D019868:Kanamycin Kinase; D010770:Phosphotransferases / Q000032:analysis*; D010947:Plants, Toxic; D011233:Precipitin Tests; D011817:Rabbits; D011994:Recombinant Proteins / Q000032:analysis; D012710:Serum Albumin, Bovine; D014026:Nicotiana / Q000032:analysis; D014162:Transfection
D003432:Cross-Linking Reagents; D007106:Immune Sera; D011994:Recombinant Proteins; D012710:Serum Albumin, Bovine; D007612:Kanamycin; D010770:Phosphotransferases; D019868:Kanamycin Kinase