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Glycosyltransferases pp 403–420Cite as

Generation of Monoclonal Antibodies to Native Active Human Glycosyltransferases

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Part of the book series: Methods in Molecular Biology ((MIMB,volume 1022))

Abstract

Complex carbohydrates serve a wide range of biological functions in cells and tissues. Their biosynthesis involves more than 200 distinct glycosyltransferases in human cells, and the expression, properties, and topology of these enzymes regulate the glycosylation patterns of proteins and lipids. Glycosyltransferases are ER-Golgi resident enzymes with slow turnover, which makes monitoring of protein expression a method more directly linked to enzyme function, than monitoring gene expression. In situ monitoring of expression and subcellular topology of glycosyltransferase proteins by immunological techniques using monoclonal antibodies therefore provides an excellent strategy to analyze the glycosylation process in cells. A major drawback has been difficulties in generating antibodies to glycosyltransferases and validating their specificities. Here we describe a simple strategy for generating and characterizing monoclonal antibodies to human glycosyltransferases. This strategy includes a process for recombinant production and purification of enzymes for immunization, a simple selection strategy for isolation of antibodies with optimal properties for in situ detection of enzyme expression, and a comprehensive strategy for characterizing the fine specificity of such antibodies.

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Acknowledgments

This work was supported by Kirsten og Freddy Johansen Fonden, A.P. Møller og Hustru Chastine Mc-Kinney Møllers Fond til Almene Formaal, The Carlsberg Foundation, The Novo Nordisk Foundation, The Danish Research Councils, a program of excellence from the University of Copenhagen, and the Danish National Research Foundation (DNRF107).

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Authors and Affiliations

  1. Department of Cellular and Molecular Medicine, Copenhagen Center for Glycomics, University of Copenhagen, Copenhagen N, Denmark

    Malene Bech Vester-Christensen, Eric Paul Bennett & Henrik Clausen

  2. Department of Odontology, Copenhagen Center for Glycomics, University of Copenhagen, Copenhagen N, Denmark

    Ulla Mandel

Authors
  1. Malene Bech Vester-Christensen
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  2. Eric Paul Bennett
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  3. Henrik Clausen
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  4. Ulla Mandel
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Editors and Affiliations

  1. Department of Medicine, Queen's University, Kingston, Ontario, Canada

    Inka Brockhausen

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Vester-Christensen, M.B., Bennett, E.P., Clausen, H., Mandel, U. (2013). Generation of Monoclonal Antibodies to Native Active Human Glycosyltransferases. In: Brockhausen, I. (eds) Glycosyltransferases. Methods in Molecular Biology, vol 1022. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-465-4_30

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  • DOI: https://doi.org/10.1007/978-1-62703-465-4_30

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  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-62703-464-7

  • Online ISBN: 978-1-62703-465-4

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