In the past decade, there has been an explosion of progress in understanding the roles of carbohydrates in biological systems. This explosive progress was made with the efforts in determining the roles of carbohydrates in immunology, neurobiology and many other disciplines, examining each unique system and employing new technology. This volume represents the second of three in the Methods in Enzymology series, including Glycobiology (vol. 415) and Functional Glycomics (vol. 417), dedicated to disseminating information on methods in determining the biological roles of carbohydrates. These books are designed to provide an introduction of new methods to a large variety of readers who would like to participate in and contribute to the advancement of glycobiology. The methods covered include structural analysis of carbohydrates, biological and chemical synthesis of carbohydrates, expression and determination of ligands for carbohydrate-binding proteins, gene expression profiling including micro array, and generation of gene knockout mice and their phenotype analyses.
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Carbohydrate-Protein Interactions-Physical Measurements
Characterization of specific donor binding to £\1,4-N-acteylhexosaminyltransferase EXTL2 using Isothermal Titration Calorimetry.
NMR Analysis of Carbohydrate-Protein Interactions.
Determination of atomic structure for glycosyltransferase-substrate complex.
Section 2 ¡V Phage Display Library
Screening of peptide-displaying phage libraries to identify short peptides mimicking carbohydrates.
Phage Display-Derived Human Antibodies Against Specific Glycosaminoglycan epitopes.
Glycosyltransferase Expression Profiling
Autofluorescent proteins for monitoring the intracellular distribution of glycosyltransferases.
Expression profiling of glycosyltransferases and their related enzymes using in situ hybridization.
Expression profiling of glycosyltransferases using RT-PCR.
Expression profiling of lycosyltransferases and related enzymes using gene micro-arrays.
CHO mutants in Glycan Biosynthesis
Lectin Resistant CHO Glycosylation Mutants.
CHO glycosylation mutants ¡V GPI anchor.
CHO glycosylation mutants ¡Vproteoglycans.
Function of Sulfation
Determination of Substrate Specificity of Sulfotransferases and Glycosyltransferases (Protoglycans).
Measuring the activities of the sulfs, two novel heparin/heparan sulfate endosulfatases.
Determination of chemokine-glycosaminoglycan interaction specificity.
Identification of Proteoglycan-binding Proteins.
Functions of glycans revealed by gene inactivation of L-selectin ligand sulfotransferases in mice.
Functions Revealed by Forced Expression or Gene Knockout
Expression of specific carbohydrates by transfection with specific carbohydrate modifying enzymes.
N-glycan structure analysis using lectins and an a-mannosidase IIx activity assay.
Targeted disruption of the gene encoding core 1 fÒ1-3-galactosyltransferase (T-synthase) causes embryonic lethality and defective angiogenesis in mice.
Roles of Mucin-Type O-glycans Synthesized by Core2B1,6-N-aetylglucosaminyltransferase.
Analysis of leukocyte rolling in vivo and in vitro.
Cell type-dependent Roles of Carbohydrates in Tumor Metastasis.