By Pierre Thibault, Susumu Honda
A suite of state-of-the-art recommendations for utilizing capillary electrophoresis (CE) to research complicated carbohydrates. those easily reproducible protocols offer tools for pattern education, research of mono- and oligosaccharides, glycoproteins, and glycoconjugates. an invaluable appendix describes the constructions of the main typically encountered carbohydrate residues and olgosaccharides from mammalian and bacterial origins. every one protocol includes precise info on reagents, equipment, notes, reviews, and pointers on techniques.
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3. Stain the PVDF membrane with amido black, cut out the protein bands of interest using a razor blade, and transfer into screw-top Eppendorf tubes. 4. 0, and 50 µL of freshly prepared 1 M sodium nitrite. 5. 5-mL Eppendorf tubes and dry. 6. Prepare 2-AB labeling reagent as described by manufacturer. 7. Take care to completely wet each blot strip with the labeling reagent (usually 15 µL are sufficient), cap the tubes, and label the strips for 2–3 h at 65°C in a heating block. 8. Wash the blot strips three times with about 10 mL of 50% acetonitrile, transfer to screw-top Eppendorf tubes, and dry.
Applications to simple mono- and oligosaccharides are described in Chapters 5 and 6, respectively. In Chapter 6 an example of an application to the analysis of N-glycans in a glycoprotein sample is also presented (16). In these chapters it is indicated that quantification is reliable, because the yields of bisPMP derivatives are quantitative. Since typical examples of analysis of monoand oligosaccharides by the plain zone electrophoresis mode and the zone electrophoresis mode as borate complexes are presented in the following chapters, examples of the separation of disaccharides by affinity electrophoresis mode (Fig.
Hart, G. , Haltiwanger, R. , Holt, G. , and Kelly, W. G. (1989) Glycosylation in the nucleus and cytoplasm. Annu. Rev. Biochem. 58, 841–874. 25. Harris, R. H. and Spellman, M. W. (1993) O-linked fucose and other post-translational modifications unique to EGF modules. Glycobiology 3, 219–224. 26. Jacob, G. S. and Scudder, P. (1994) Glycosidases in structural analysis. Methods Enzymol. 230, 280–299. 27. Merry, A. , and Ioannides, A. (1992) Automated simultaneous release of intact and unreduced N- and O-linked glycans from glycoproteins.