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
*These authors contributed equally to this work.
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- 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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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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