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Full Description
This volume presents detailed, recently-developed protocols ranging from isolation of nuclei to purification of chromatin regions containing single genes, with a particular focus on some less well-explored aspects of the nucleus. The methods described include new strategies for isolation of nuclei, for purification of cell type-specific nuclei from a mixture, and for rapid isolation and fractionation of nucleoli. For gene delivery into and expression in nuclei, a novel gentle approach using gold nanowires is presented. As the concentration and localization of water and ions are crucial for macromolecular interactions in the nucleus, a new approach to measure these parameters by correlative optical and cryo-electron microscopy is described. The Nucleus, Second Edition presents methods and software for high-throughput quantitative analysis of 3D fluorescence microscopy images, for quantification of the formation of amyloid fibrils in the nucleus, and for quantitative analysis of chromosome territory localization. Written in the successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible protocols, and notes on troubleshooting and avoiding known pitfalls.
Authoritative and easily accessible, The Nucleus, Second Edition seeks to serve both professionals and novices with its well-honed methods for the study of the nucleus.
Contents
Cell Type-Specific Affinity Purification of Nuclei for Chromatin Profiling in Whole Animals.- Lysis Gradient Centrifugation - A Flexible Method for the Isolation of Nuclei from Primary Cells.- Isolation of Nuclei in Media Containing an Inert Polymer to Mimic the Crowded Cytoplasm.- A New Rapid Method for Isolating Nucleoli.- Sequential Recovery of Macromolecular Components of the Nucleolus.- Au Nanoinjectors for Electrotriggered Gene Delivery into the Cell Nucleus.- Improving Chromatin Immunoprecipitation (ChIP) by Suppression of Method-Induced DNA-Damage Signaling.- Purification of Specific Chromatin Loci for Proteomic Analysis.- Chromatin Structure Analysis of Single Gene Molecules by Psoralen Crosslinking and Electron Microscopy.- Purification of Proteins on Newly Synthesized DNA Using iPOND.- Applying the Ribopuromycylation Method to Detect Nuclear Translation.- Targeted Nano Analysis of Water and Ions in the Nucleus Using Cryo-Correlative Microscopy.- A Redox-Sensitive Yellow Fluorescent Protein Sensor for Monitoring Nuclear Glutathione Redox Dynamics.- Determination of the Dissociation Constant of the NFkB p50/p65 Heterodimer in Living Cells Using Fluorescence Cross-Correlation Spectroscopy.- Imaging and Quantification of Amyloid Fibrillation in the Cell Nucleus.- Analysis of Nuclear Organization with TANGO, Software for High-Throughput Quantitative Analysis of 3D Fluorescence Microscopy Images.- Quantitative Analysis of Chromosome Localization in the Nucleus.
