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A new sensitive method for qualitative and quantitative assay of neomycin phosphotransferase in crude cell extracts

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Sprengel,  Rolf
Department of Molecular Neurobiology, Max Planck Institute for Medical Research, Max Planck Society;
Rolf Sprengel Group, Max Planck Institute for Medical Research, Max Planck Society;
Olfaction Web, Max Planck Institute for Medical Research, Max Planck Society;

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https://www.sciencedirect.com/science/article/pii/0378111984901227/pdf?md5=f96ab2907134f7ebc9a5e2ba103bb421&pid=1-s2.0-0378111984901227-main.pdf
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https://doi.org/10.1016/0378-1119(84)90122-7
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Citation

Reiss, B., Sprengel, R., Will, H., & Schaller, H. (1984). A new sensitive method for qualitative and quantitative assay of neomycin phosphotransferase in crude cell extracts. Gene, 30(1-3), 211-217. doi:10.1016/0378-1119(84)90122-7.


Cite as: https://hdl.handle.net/21.11116/0000-0000-D3C3-5
Abstract
A general method is described for the detection and quantification of low amounts of neomycin phosphotransferase in crude cell extracts. The assay is based on the electrophoretic separation of the enzyme from other interfering proteins and detection of its enzymatic activity by in situ phosphorylation of the antibiotic kanamycin. Both kanamycin and [gamma-32P]ATP acting as substrates are embedded in an agarose gel placed on the polyacrylamide gel containing the separated proteins. After the enzymatic reaction, the phosphorylated kanamycin is transferred to P81 phosphocellulose ion exchange paper and the radiolabeled kanamycin is visualised by autoradiography. With this method 1 ng of active enzyme can easily be detected. Both prokaryotic and eukaryotic cell extracts can be examined, and changes in the size of enzymatically active proteins can be determined.