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  Recombinant ganglioside GM2-synthase-expression in insect cells and enzyme assay

Wendeler, M., Reiländer, H., Hoernschemeyer, J., Schwarzmann, G., Kolter, T., & Sandhoff, K. (2003). Recombinant ganglioside GM2-synthase-expression in insect cells and enzyme assay. In Yuan C. Lee, & Reiko T. Lee (Eds.), Methods in Enzymology (pp. 476-489). Elsevier Inc. doi:10.1016/S0076-6879(03)01073-5.

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Datensatz-Permalink: https://hdl.handle.net/11858/00-001M-0000-0024-DB93-A Versions-Permalink: https://hdl.handle.net/21.11116/0000-0007-1DA2-2
Genre: Buchkapitel

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 Urheber:
Wendeler, Michaela, Autor
Reiländer, Helmut1, Autor           
Hoernschemeyer, Jörg, Autor
Schwarzmann, Günter, Autor
Kolter, Thomas, Autor
Sandhoff, Konrad, Autor
Affiliations:
1Department of Molecular Membrane Biology, Max Planck Institute of Biophysics, Max Planck Society, ou_2068290              

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Schlagwörter: -
 Zusammenfassung: Publisher Summary:
This chapter investigates the recombinant ganglioside GM2 synthase; and describes the expression of murine β-1,4-N-acetylgalactosaminyltransferase both as a complete membrane-bound enzyme and as a soluble form in the baculovirus insect -cell expression system. A novel assay based on radiochemically labeled GM3 prepared from the tissue ganglioside, which permits the evaluation of potential UDP-GalNAc analogs is presented. Schematic protein structure of GM2 synthase showing the topology of type II transmembrane proteins is presented. For the expression of full-length GM2 synthase, the entire cDNA of GM2 synthase in the vector pCMV Blue is used as a template in the polymerase chain reaction (PCR) reaction. For expression of soluble GM2 synthase, the medium of infected cells is harvested 72 h postinfection by centrifugation (2000 rpm, 10 min) and concentrated 15-fold with a Vivaspin 4-ml concentrator with a PES membrane and 30,000 moleculor weight cut off (MWCO). This concentrated supernatant is prepared fresh for the assays and is always kept on ice. The chapter discusses the immunoblot analysis, standard glycosyltransferase assays, and kinetic analysis of recombinant GM2 synthase.

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Sprache(n): eng - English
 Datum: 2004-01-072003
 Publikationsstatus: Erschienen
 Seiten: 14
 Ort, Verlag, Ausgabe: -
 Inhaltsverzeichnis: -
 Art der Begutachtung: Expertenbegutachtung
 Identifikatoren: DOI: 10.1016/S0076-6879(03)01073-5
 Art des Abschluß: -

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Titel: Methods in Enzymology
  Untertitel : Recognition of Carbohydrates in Biological Systems, Part B: Specific Applications
Genre der Quelle: Reihe
 Urheber:
Yuan C. Lee, Herausgeber
Reiko T. Lee, Herausgeber
Affiliations:
-
Ort, Verlag, Ausgabe: Elsevier Inc
Seiten: 625 Band / Heft: 363 Artikelnummer: - Start- / Endseite: 476 - 489 Identifikator: ISBN: 978-0-12-182266-8