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A proven collection of readily reproducible techniques for studying amyloid proteins and their involvement in the etiology, pathogenesis, diagnosis, and therapy of amyloid diseases. The contributors provide methods for the preparation of amyloid and its precursors (oligomers and protofibrils), in vitro assays and analytical techniques for their study, and cell culture models and assays for the production of amyloid proteins. Additional chapters present readily reproducible techniques for amyloid extraction from tissue, its detection in vitro and in vivo, as well as nontransgenic methods for developing amyloid mouse models. The protocols follow the successful Methods in Molecular Biology(TM) series format, each offering step-by-step laboratory instructions, an introduction outlining the principle behind the technique, lists of the necessary equipment and reagents, and tips on troubleshooting and avoiding known pitfalls.
List of contents
In Vitro Assays.- Preparation of Aggregate-Free,Low Molecular Weight Amyloid? for Assembly and Toxicity Assays.- Determination of Peptide Oligomerization State Using Rapid Photochemical Crosslinking.- In Vitro Preparation of Prefibrillar Intermediates of Amyloid-? and ? Synuclein.- Purification of Recombinant Tau Protein and Preparation of Alzheimer-Paired Helical Filaments In Vitro.- Cyclic Amplification of Protein Misfolding and Aggregation.- X-Ray Diffraction Studies of Amyloid Structure.- Molecular Electron Microscopy Approaches to Elucidating the Mechanisms of Protein Fibrillogenesis.- Time -Lapse Atomic Force Microscopy in the Characterization of Amyloid-Like Fibril Assembly and Oligomeric Intermediates.- Fourier Transform Infrared and Circular Dichroism Spectroscopies for Amyloid Studies.- Quasielastic Light Scattering for Protein Assembly Studies.- Intrinsic Fluorescent Detection of Tau Conformation and Aggregation.- Quantitative Measurement of Fibrillogenesis by Mass Spectrometry.- Cell Culture Assays.- Isolation and Culturing of Human Vascular Smooth Muscle Cells.- Murine Cerebrovascular Cells as a Cell Culture Model for Cerebral Amyloid Angiopathy.- Purification of Human Wild-Type or Variant Cystatin C From Conditioned Media of Transfected Cells.- Prion Propagation in Cell Culture.- In Vivo-Related Assays.- Preparation and Propagation of Amyloid-Enhancing Factor.- Purification of Amyloid Protein AA Subspecies From Amyloid-Rich Human Tissues.- Purification of Transthyretin and Transthyretin Fragments From Amyloid-Rich Human Tissues.- Extraction and Chemical Characterization of Tissue-Deposited Proteins From Minute Diagnostic Biopsy Specimens.- Tissue Processing Prior to Protein Analysis and Amyloid-ß Quantitation.- ELISA Method for Measurement ofAmyloid-ß Levels.- Histological Staining of Amyloid-ß in Mouse Brains.- The Mouse Model for Scrapie.- Radiolabeling of Amyloid-ß Peptides.- In Vivo Imaging of Amyloid-ß Deposits in Mouse Brain With Multiphoton Microscopy.- Magnetic Resonance Imaging of Amyloid Plaques in Transgenic Mice.
Summary
A proven collection of readily reproducible techniques for studying amyloid proteins and their involvement in the etiology, pathogenesis, diagnosis, and therapy of amyloid diseases. The contributors provide methods for the preparation of amyloid and its precursors (oligomers and protofibrils), in vitro assays and analytical techniques for their study, and cell culture models and assays for the production of amyloid proteins. Additional chapters present readily reproducible techniques for amyloid extraction from tissue, its detection in vitro and in vivo, as well as nontransgenic methods for developing amyloid mouse models. The protocols follow the successful Methods in Molecular Biology™ series format, each offering step-by-step laboratory instructions, an introduction outlining the principle behind the technique, lists of the necessary equipment and reagents, and tips on troubleshooting and avoiding known pitfalls.
