Hancock R. (Ed.) The Nucleus

2nd Edition. — Humana Press, 2015. — 236 p. — ISBN: 9781493916795
Series: Methods in Molecular Biology, № 1228This 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.Isolation of Nuclei
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
Nucleoli
A New Rapid Method for Isolating Nucleoli
Sequential Recovery of Macromolecular Components of the Nucleolus
Genes and Chromatin
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 Cross-Linking and Electron Microscopy
Purification of Proteins on Newly Synthesized DNA Using iPOND
Applying the Ribopuromycylation Method to Detect Nuclear Translation
The Intranuclear Milieu
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
Imaging Nuclear Structures
Determination of the Dissociation Constant of the NFκB 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