Journal
NATURE PROTOCOLS
Volume 5, Issue 5, Pages 821-833Publisher
NATURE PUBLISHING GROUP
DOI: 10.1038/nprot.2010.17
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Funding
- Biotechnology & Biological Sciences Research Council [6E/16799, B65/8/02649]
- Engineering & Physical Sciences Research Council [GR/S79268/01, EP/G037604]
- Wellcome Trust [082502]
- Medical Research Council
- BBSRC [BB/D006325/1] Funding Source: UKRI
- MRC [G117/423] Funding Source: UKRI
- Biotechnology and Biological Sciences Research Council [BB/D006325/1] Funding Source: researchfish
- Engineering and Physical Sciences Research Council [GR/S79268/01] Funding Source: researchfish
- Medical Research Council [G117/423] Funding Source: researchfish
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Natural and semi-synthetic heparan sulfate (HS) saccharide libraries are a valuable resource for investigating HS structure-function relationships, enabling high-throughput glycomics studies. Owing to the difficulty of chemical or in vitro enzymatic synthesis of HS saccharides, the structural diversity displayed in saccharides from tissue or cell sources cannot be readily accessed. In contrast, saccharide libraries can be generated by partial digestion of tissue-derived HS polysaccharide chains and chromatographic fractionation of the resulting saccharide mixtures. Fractionation is initially on the basis of hydrodynamic volume, using size exclusion chromatography. Further fractionation, on the basis of charge using strong anion exchange, can subsequently be applied. Desalting and sample concentration follows each fractionation step. Chromatographic fractions are generated that contain purified, or partially purified, saccharides. Here we describe a comprehensive protocol for generation of structurally diverse natural saccharide libraries from HS variants that is fast (similar to 3 weeks) and reproducible.
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