A sensitive and simple assay for neomycin phosphotransferase II activity in transgenic tissue.
A new assay for the detection of the enzyme neomycin phosphotransferase II (NPTII) in crude cell extracts is described. The method is based on the chromatographic separation of the compounds resulting from the reaction of NPTII with kanamycin (Km) and [gamma-32P]ATP; the labelled Km.phosphate is subsequently detected by autoradiography. Chromatography is carried out on polyethyleneimine cellulose plates. This assay has been tested with bacterial, plant and animal crude extracts and comparisons with the assays in current use have been made. Our assay has several advantages: it is simple, rapid, sensitive and safe.
Cabanes-Bastos
E
E
Centre for Biotechnology, Imperial College of Science, Technology and Medicine, London, U.K.
Day
A G
AG
Lichtenstein
C P
CP
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D030801:Animals, Genetically Modified; D001345:Autoradiography; D002855:Chromatography, Thin Layer / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology; D007612:Kanamycin / Q000494:pharmacology; D019868:Kanamycin Kinase; D008722:Methods; D010770:Phosphotransferases / Q000032:analysis* / Q000235:genetics; D010944:Plants / Q000201:enzymology / Q000235:genetics; D011094:Polyethyleneimine; D012680:Sensitivity and Specificity; D013379:Substrate Specificity
D007612:Kanamycin; D000255:Adenosine Triphosphate; D011094:Polyethyleneimine; D010770:Phosphotransferases; D019868:Kanamycin Kinase