Beschreibung
Proteoglycans are some of the most elaborate macromolecules of mammalian and lower organisms. The covalent attachment of at least five types of glycosami- glycan side chains to more than forty individual protein cores makes these molecules quite complex and endows them with a multitude of biological functions. Proteoglycan Protocols offers a comprehensive and up-to-date collection of prepa- tive and analytical methods for the in-depth analysis of proteoglycans. Featuring st- by-step detailed protocols, this book will enable both novice and experienced researchers to isolate intact proteoglycans from tissues and cultured cells, to establish the composition of their carbohydrate moieties, to generate strategies for prokaryotic and eukaryotic expression, to utilize methods for the suppression of specific proteoglycan gene expression and for the detection of mutant cells and degradation products, and to study specific interactions between proteoglycans and extracellular matrix proteins as well as growth factors and their receptors. The readers will find concise, yet comprehensive techniques carefully drafted by leading experts in the field. Each chapter commences with a general Introduction, followed by a detailed Materials section, and an easy-to-follow Methods section. An asset of each chapter is the extensive notation that includes troubleshooting tips and practical considerations that are often lacking in formal methodology papers. The reader will find this section most valuable because it is clearly provided by experienced scientists who have first-hand knowledge of the techniques they outline. In addition, most of the chapters are well illustrated with examples of typical data generated with each method.
Inhaltsverzeichnis
I. Isolation and Purification Isolation of Proteoglycans from Cell Cultures and Tissues Masaki Yanagishita Isolation of Proteoglycans from Tendon Kathryn G. Vogel and Julie A. Peters Purification of Proteoglycans from Mineralized Tissues Neal S. Fedarko Purification of Perlecan from Endothelial Cells John Whitelock Isolation and Characterization of Nervous Tissue Proteoglycans Yu Yamaguchi Analysis of Proteoglycans and Glycosaminoglycans from Drosophila William D. Staatz, Hidenao Toyoda, Akiko Kinoshita-Toyoda, Kimberlly Chhor, and Scott B. Selleck Cartilage and Smooth Muscle Cell Proteoglycans Detected by Affinity Blotting Using Biotinylated Hyaluronan James Melrose Preparation of Proteoglycans for N-Terminal and Internal Amino Acid Sequence Analysis Peter J. Neame High-Specific-Activity 35S-Labeled Heparan Sulfate Prepared from Cultured Cells Nicholas W. Shworak Selective Detection of Sulfotransferase Isoforms by the Ligand Affinity-Conversion Approach Nicholas W. Shworak Particulate and Soluble Glycosaminoglycan-Synthesizing Enzymes: Preparation Assay and Use Geetha Sugumaran and Jeremiah E. Silbert Disaccharide Composition of Hyaluronan and Chondroitin/Dermatan Sulfate: Analysis with Fluorophore-Assisted Carbohydrate Electrophoresis Anna H. K. Plaas, L. West, Ronald J. Midura, and Vincent C. Hascall Integral Glycan Sequencing of Heparan Sulfate and Heparin Saccharides Jeremy E. Turnbull Analytical and Preparative Strong Anion-Exchange HPLC of Heparan Sulfate and Heparin Saccharides Jeremy E. Turnbull Proteoglycans Analyzed by Composite Gel Electrophoresis and Immunoblotting George R. Dodge and Ralph Heimer Quantitation of Proteoglycans in Biological Fluids Using Alcian Blue Madeleine Karlsson and Sven Björnsson Cellulose Acetate Electrophoresis of Glycosaminoglycans Yanusz Wegrowski and Francois-Xavier Maquart Disaccharide Composition in Glycosaminoglycans/Proteoglycans Analyzed by Capillary Zone Electrophoresis Nikos K. Karamanos and Anders Hjerpe Intact and Oligomeric Glycosaminoglycans Analyzed with Capillary Electrophoresis Nikos K. Karamanos and Anders Hjerpe II. Expression, Detection, and Degradation Recombinant Expression of Proteoglycans in Mammalian Cells: Utility and Advantages of the Vaccinia Virus/T7 Bacteriophage Hybrid Expression System David J. McQuillan, Neung-Seon Seo, Anne M. Hocking, and Camille I. McQuillan Purification of Recombinant Human Decorin and Its Subdomains Anna L. McBain and David M. Mann Prokaryotic Expression of Proteoglycans Alan D. Murdoch and Renato V. Iozzo Proteoglycan Gene Targeting in Somatic Cells Giancarlo Ghiselli and Renato V. Iozzo Constitutive and Inducible Antisense Expression Melissa Handler and Renato V. Iozzo Cell-Mediated Transfer of Proteoglycan Genes Jens W. Fischer, Michael G. Kinsella, David Hasenstab, Alexander W. Clowes, and Thomas N. Wight Morphological Evaluation of Proteoglycans in Cells and Tissues Stephanie L. Lara, Stephen P. Evanko, and Thomas N. Wight Expression and Characterization of Engineered Proteoglycans Wei Luo, Chunxia Guo, Jing Zheng, and Marvin L. Tanzer Intracellular Localization of Engineered Proteoglycans Tung-Ling L. Chen, Peiyin Wang, Seung S. Gwon, Nina W. Flay, and Barbara M. Vertel Selection of Glycosaminoglycan-Deficient Mutants Xiaomei Bai, Brett Crawford, and Jeffrey D. Esko
