By Pierre Thibault, Susumu Honda
A suite of state-of-the-art strategies for utilizing capillary electrophoresis (CE) to research advanced carbohydrates. those easily reproducible protocols supply tools for pattern practise, research of mono- and oligosaccharides, glycoproteins, and glycoconjugates. an invaluable appendix describes the constructions of the main normally encountered carbohydrate residues and olgosaccharides from mammalian and bacterial origins. every one protocol comprises distinct details on reagents, gear, notes, reviews, and pointers on strategies.
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14. , Trimble, R. , Tarentino, A. L. and Plummer, T. H. Jr. (1989) Characterization of glycoproteins and their associated oligosaccharides through the use of endoglycosidases. Analyt. Biochem. 180, 195. 40 Merry and Astrautsova 15. Tarentino, A. L. and Maley, F. (1975) A comparison of the substrate specificities of endo-beta-N-acetylglucosaminidases from Streptomyces griseus and Diplococcus pneumoniae. Biochem. Biophys. Res. Commun. 67, 455–462. 16. , and Kobata, A. (1977) Structures of the carbohydrate moiety of ovalbumin glycopeptide III and the difference in specificity of endo-beta-N-acetylglucosaminidases CII and H.
6. Store remaining glycoprotein at 4°C for future use. 7. 4% SDS. 8. Incubate for 3 min at 100°C. 9. Cool and add 50 µL of CHAPS detergent buffer. 10. 2 U (2 µL) of PNGase F. 11. Incubate for 24 h at 37°C (add 5 µL of toluene to prevent bacterial growth). 12. Remove 5 µL and analyze the reaction mixture by SDS-PAGE (see Note 11). 13. If sample is completely deglycosylated proceed with step 14 otherwise continue with incubation. 14. Filter samples through protein binding membrane or perform gel filtration (see Note 12).
2. CE: Use any apparatus equipped with a high-voltage power supply, a sample injection device, and a laser-induced fluorescence detector. Make a detection window on a capillary by burning a short segment (3–4 mm) of the polyimide coating and mount the capillary on the capillary holder. 1 M sodium hydroxide and rinse it with the running buffer (see Note 22) for 2 min each. Detect the separated derivatives at approx 520 nm with irradiation by He–Cd laser. 3. The NBD-MG Method 1. Release of N-glycans from glycoproteins (see Note 23): Add 10 µL of a 5% w/v SDS solution in 10 w/v% aqueous solution of 2-mercaptoethanol to a glycoprotein sample (~100 µg) and heat the mixture for 5 min on a boiling water bath for denaturation of the glycoprotein sample.