A Laboratory Guide to Glycoconjugate Analysis

18. 2 Principle of FACE/Gel Retardation Assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 349 18. 3 Labelling of Oligosaccharides with ANTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 350 18. 4 Screening of Carbohydrate Ligands for Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . 352 18. 5 Measurement of Binding Constant for the Interaction Between Protein and ANTS-Labelled Carbohydrate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ... Read More

18. 2 Principle of FACE/Gel Retardation Assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 349 18. 3 Labelling of Oligosaccharides with ANTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 350 18. 4 Screening of Carbohydrate Ligands for Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . 352 18. 5 Measurement of Binding Constant for the Interaction Between Protein and ANTS-Labelled Carbohydrate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 355 18. 6 Measurement of Binding Constant for the Interaction Between Protein and Native Carbohydrate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 357 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 360 ~ The Application of Capillary Affinity Electrophoresis to the Analysis _ of Carbohydrate-Protein Interactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 361 19. 1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 361 19. 2 Principle of CAE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 363 19. 3 Determination of Association Constants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 364 19. 4 Technical Procedures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 366 General considerations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 366 19. 5 Limitations of the Technique . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . 370 19. 6 Application of CAE to the Analysis of Carbohydrate-Protein Interactions . . . . . . 371 19. 7 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 375 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 377 20. 1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 379 Definitions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 380 20. 2 Technical Procedures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 381 20. 3 Sample Detection and Sample Recovery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 389 Autoradiography and staining . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 389 Sample detection by blotting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 389 Semipreparative ACE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 390 20. 4 Analysis of Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 391 Measuring sample mobilities - calculating a retardation coefficient . . . . . . . . . . . . 391 Graphical analysis of data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 392 Interpreting ACE patterns . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 393 Reverse ACE . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 395 20. 5 Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 397 Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 398 References . . . . . . . Read Less

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