By Gerald W. Hart (auth.), Pierre Thibault, Susumu Honda (eds.)
Recent advancements in high-resolution separation suggestions in accordance with capillary-scale chromatography and electrophoresis have reworked the research of loose and conjugated mono- and oligosaccharides. In Capillary Electrophoresis of Carbohydrates, hands-on specialists describe state-of-the-art strategies in capillary electrophoresis (CE) for the research of advanced carbohydrates. Written in step by step element to make sure profitable experimental effects, those comfortably reproducible protocols supply equipment for pattern instruction and research of mono- and oligosaccharides, glycoproteins, and glycoconjugates. diverse CE separation codecs, together with capillary area electrophoresis (CZE), capillary isoelectric focusing (CIEF), capillary isotachophoresis (CITP), capillary gel electrophoresis (CGE), and micellar electrokinetic chromatography (MEKC), are offered for various varieties of functions, corresponding to the research of glycoforms, the separation of enantiomers, and the answer of oligosaccharides. The separation of local derivatized carbohydrates is gifted utilizing UV, fluorescence, and mass spectrometric detection. different purposes exhibit using CE as an assay procedure for tracking glycosyltransferase job and for making a choice on the organization consistent of carbohydrate-protein interplay. an invaluable appendix describes the constructions of the main regularly encountered carbohydrate residues and oligosaccharides from mammalian and bacterial origins. each one protocol comprises distinct details on reagents, equipment, and useful reviews and notes on methods.
well timed and hugely sensible, Capillary Electrophoresis of Carbohydrates presents either amateur and skilled CE analysts with robust instruments for the winning separation of complicated carbohydrates by way of capillary electrophoresis.
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Extra resources for Capillary Electrophoresis of Carbohydrates
If protein precipitates) and in these situations purification by reverse-phase chromatography in volatile solvents that may be removed under vacuum is recommended. 16. Protein samples for hydrazinolysis should be essentially salt free. Salts, heavy metal ions, dyes, and detergents may interfere with the hydrazinolysis reaction in an unpredictable way and need to be removed. Unless the sample has been desalted by other techniques such as reverse-phase chromatography (see Note 15) this is most conveniently performed by dialysis.
Harvey, D. , Wormald, M. , and Parekh, R. B. (1994) Analysis of glycoprotein-associated oligosaccharides. Annu. Rev. Biochem. 62, 65–100. 7. , Wheeler, S. , Hunter, A. , Dwek, R. , and Harvey, D. J. (1997) Sequencing of N-linked oligosaccharides directly from protein gels: in-gel deglycosylation followed by matrix-assisted laser desorption/ionization mass spectrometry and normal-phase high-performance liquid chromatography. Anal. Biochem. 250, 82–101. 8. Rudd, P. , Guile, G. , Harvey, D. , and Dwek, R.
Deglycosylatation can also be performed using PNGase A. But in this case the protein should be cleaved to glycopeptides by appropriate protease enzymes. This is especially relevant if plant glycoproteins are being studied, as PNGase F will not cleave oligosaccharides with core fucose residues in α1–3 linkage. 14. 8 kDa. If a 2-mL column has an exclusion volume of about 600 µL, then elution of the column with 800 µL of water should elute all N-linked oligosaccharides. If in doubt, fractions can be assayed for hexose using the phenol–sulfuric acid method.
Capillary Electrophoresis of Carbohydrates by Gerald W. Hart (auth.), Pierre Thibault, Susumu Honda (eds.)