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

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.

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Item Permalink: http://hdl.handle.net/21.11116/0000-0000-D3C3-5 Version Permalink: http://hdl.handle.net/21.11116/0000-0000-D3C4-4
Genre: Journal Article

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Gene_30_1984_211.pdf (Any fulltext), 664KB
 
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 Creators:
Reiss, Bernd, Author
Sprengel, Rolf1, 2, 3, Author              
Will, Hans, Author
Schaller, Heinz, Author
Affiliations:
1Department of Molecular Neurobiology, Max Planck Institute for Medical Research, Max Planck Society, ou_1497704              
2Rolf Sprengel Group, Max Planck Institute for Medical Research, Max Planck Society, ou_1497741              
3Olfaction Web, Max Planck Institute for Medical Research, Max Planck Society, ou_1497733              

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Free keywords: Recombinant DNA; kanamycin resistance gene; enzymatic assay; nondenaturing gel electrophoresis; in situ phosphorylation; detection of altered proteins
 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.

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Language(s): eng - English
 Dates: 1984-05-101984-03-291984-05-102003-02-041984-10-01
 Publication Status: Published in print
 Pages: 7
 Publishing info: -
 Table of Contents: -
 Rev. Type: Peer
 Degree: -

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Title: Gene
  Other : Gene
Source Genre: Journal
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Publ. Info: Amsterdam : Elsevier
Pages: - Volume / Issue: 30 (1-3) Sequence Number: - Start / End Page: 211 - 217 Identifier: ISSN: 0378-1119
CoNE: https://pure.mpg.de/cone/journals/resource/954925526821