A Laboratory Guide to Glycoconjugate Analysis by Uwe D. Neue, Charles H. Phoebe (auth.), Dr. Peter Jackson,

By Uwe D. Neue, Charles H. Phoebe (auth.), Dr. Peter Jackson, Prof. John T. Gallagher (eds.)

18. 2 precept of FACE/Gel Retardation Assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 349 18. three Labelling of Oligosaccharides with ANTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 350 18. four Screening of Carbohydrate Ligands for Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . 352 18. five size of Binding consistent for the interplay among Protein and ANTS-Labelled Carbohydrate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 355 18. 6 dimension of Binding consistent for the interplay among Protein and local Carbohydrate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 357 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 360 ~ the applying of Capillary Affinity Electrophoresis to the research _ of Carbohydrate-Protein Interactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 361 19. 1 advent . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 361 19. 2 precept of CAE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 363 19. three choice of organization Constants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 364 19. four Technical strategies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 366 normal issues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 366 19. five obstacles of the method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 370 19. 6 software of CAE to the research of Carbohydrate-Protein Interactions . . . . . . 371 19. 7 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 375 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 377 20. 1 advent . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 379 Definitions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 380 20. 2 Technical techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 381 20. three pattern Detection and pattern restoration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 389 Autoradiography and marking . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 389 pattern detection by means of blotting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 389 Semipreparative ACE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 390 20. four research of knowledge . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 391 Measuring pattern mobilities - calculating a retardation coefficient . . . . . . . . . . . . 391 Graphical research of knowledge . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 392 reading ACE styles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 393 opposite ACE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 395 20. five precis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 397 Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 398 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 398 topic Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 399 XII record of individuals Nebojsa Avdalovic John T. Gallagher Dionex company melanoma study crusade division of clinical Oncology 445 Lakeside force college of Manchester Sunnyvale, CA 94086 Christie CRC study Centre Klaus Biemann Wilmslow highway division of Chemistry Manchester M20 4BX Massachusetts Institute of know-how united kingdom Cambridge, MA 02139-4307 united states Geoffrey R.

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Native and biotinylated molecular weight markers are also provided for use with the protocols as described above. • Any suitable format of SDS gel electrophoresis equipment, such as the Bio-Rad minigel system or the Pharmacia Phastgel system. g. g. following SDS-PAGE (10), Western blotting (11, 12) or dot blotting. g. PhastTransfer™ Pharmacia). g. PVDF, nitrocellulose and nylon, may be used. Although the streptavidin-alkaline phosphatase detection system will function with nylon membranes, high backgrounds are observed.

G. ovalbumin) in the same buffer solution as the sample to discover to what extent the buffer has interfered with the reaction. 2. 5 mglml in water) to the samples, mix thoroughly and incubate for 20 min in the dark, at room temperature. 3. Stop the oxidation reaction by adding of 5 III of sodium bisulphite solution (10 mglml in 200 mM sodium periodate), mix thoroughly and incubate at room temperature for 5 min. 4. Perform biotinylation by addition of the hydrazide solution, mix and incubate for 1 hat room temperature.

It therefore may limit its use in further experiments, for example in enzyme work. 1 summarizes a few of the currently available precolumn derivatization techniques. They are all variants of the general scheme of reductive amination. Some are covered in more detail in other parts of the book. For additional reactions, see ref. 54. 1 Precolumn derivatization via reductive amination Reagent and Detector Detection Comments References Signal, Oxford 52,55 limits reaction conditions 2-aminobenzamide, Fluorescence, 2 h at 65°C (excitation 330 nm, 5-10pmol Glycosystems emission 420 nm) 2-aminobenzoic acid, Fluorescence, 20 20 min at 80°C 55,56 100 fmol (excitation 230 nm, emission 425 nm) 2-aminopyridine, Fluorescence, 13 min at 100°C (excitation 320 nm, pmol range Takara 57 Biochemical emission 400 nm) 2-aminopyridine, same as same as sialic acid residues 40, 58 50 min at 80°C above above are not released p-aminobenzoic UVNlS 59,60 ethylester, 30 min at 80 °c A different scheme is labelling with I-phenyl-3-methyl-5-pyrazolone (49) or 1-(pmethoxy)phenyl-3-methyl-5-pyrazolone (61).

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