A new sensitive method for qualitative and quantitative assay of neomycin phosphotransferase in crude cell extracts.
A new sensitive method for qualitative and quantitative assay of neomycin phosphotransferase in crude cell extracts.
复制标题
一种新的灵敏方法，用于定性和定量测定粗细胞提取物中的新霉素磷酸转移酶。
DOI：
10.1016/0378-1119(84)90122-7
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发表时间：
1984
期刊：
影响因子：
3.5
通讯作者：
Heinz Schaller
中科院分区：
文献类型：
--
作者：
Bernd Reiss;R. Sprengel;Hans Will;Heinz Schaller
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 [γ32P]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.
