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A Rapid Procedure for the Purification of 8-Aminopyrene Trisulfonate (APTS)-Labeled Glycans for Capillary Electrophoresis (CE)-Based Enzyme Assays

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Protein-Carbohydrate Interactions

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1588))

Abstract

Purified glycan standards are required for glycan arrays, characterizing substrate specificities of glycan-active enzymes, and to serve as retention-time or mobility standards for various separation techniques. This chapter describes a method for the rapid separation, and subsequent desalting, of glycans labeled with the highly fluorescent fluorophore 8-aminopyrene 1,3,6-trisulfonate (APTS). By using fluorophore-assisted carbohydrate electrophoresis (FACE) on polyacrylamide gels, which utilizes equipment readily available in most molecular biology laboratories, many APTS-labeled glycans can be simultaneously resolved. Excising specific gel bands containing the desired APTS-labeled glycans, followed by glycan elution from the gel and subsequent solid-phase extraction (SPE), yields single glycan species free of excess labeling reagents and buffer components. This chapter describes a FACE/SPE procedure ideal for preparing glycans for capillary electrophoresis (CE)-based enzyme assays, as well as for the purification of rare, commercially unavailable glycans from tissue culture samples.

*These authors contributed equally to this work.

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Abbreviations

APTS:

8-Aminopyrene-1,3,6-trisulfonate

CE:

Capillary electrophoresis

SPE:

Solid-phase extraction

FACE:

Fluorophore-assisted carbohydrate electrophoresis

PGC:

Porous graphitized carbon

ACN:

Acetonitrile

TFA:

Trifluoroacetic acid

PNGaseF:

Peptide N-glycosidase F

AcOH:

Acetic acid

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Acknowledgments

We thank Dr. David Rose for providing the ER glucosidase I and the National Sciences and Engineering Research Council of Canada (NSERC) for funding the CE-LIF.

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Author notes

    Authors and Affiliations

    1. Simon Fraser University, Department of Molecular Biology and Biochemistry, 8888 University Drive, Burnaby, BC, Canada, V5A 1S6

      Hayden J. Danyluk

    2. Department of Chemistry, The University of British Columbia, Okanagan, Kelowna, BC, Canada

      Leona K. Shum & Wesley F. Zandberg

    3. Department of Chemistry, Science Building, 1177 Research Road, Kelowna, BC, Canada, V1M 1V7

      Wesley F. Zandberg

    Authors
    1. Hayden J. Danyluk
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    2. Leona K. Shum
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    3. Wesley F. Zandberg
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    Corresponding author

    Correspondence to Wesley F. Zandberg .

    Editor information

    Editors and Affiliations

    1. Agriculture and Agri-Food Canada, Lethbridge, Alberta, Canada

      D. Wade Abbott

    2. Biotechnology Institute, University of Groningen, Groningen, The Netherlands

      Alicia Lammerts van Bueren

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    Danyluk, H.J., Shum, L.K., Zandberg, W.F. (2017). A Rapid Procedure for the Purification of 8-Aminopyrene Trisulfonate (APTS)-Labeled Glycans for Capillary Electrophoresis (CE)-Based Enzyme Assays. In: Abbott, D., Lammerts van Bueren, A. (eds) Protein-Carbohydrate Interactions. Methods in Molecular Biology, vol 1588. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6899-2_18

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    • DOI: https://doi.org/10.1007/978-1-4939-6899-2_18

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    • Publisher Name: Humana Press, New York, NY

    • Print ISBN: 978-1-4939-6898-5

    • Online ISBN: 978-1-4939-6899-2

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