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

  • Hayden J. Danyluk
  • Leona K. Shum
  • Wesley F. Zandberg
Protocol
Part of the Methods in Molecular Biology book series (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.

Key words

Glycan-purification Capillary electrophoresis (CE) Solid-phase extraction (SPE) Graphite Fluorophore-assisted carbohydrate electrophoresis (FACE) 

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

Notes

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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Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  • Hayden J. Danyluk
    • 1
  • Leona K. Shum
    • 2
  • Wesley F. Zandberg
    • 2
    • 3
  1. 1.Simon Fraser UniversityDepartment of Molecular Biology and BiochemistryBurnabyCanada
  2. 2.Department of ChemistryThe University of British Columbia, OkanaganKelownaCanada
  3. 3.Department of ChemistryKelownaCanada

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