The Nucleus

ISBN-13: 9781493916795 / Angielski / Twarda / 2014 / 237 str.

Ronald Hancock

Opis

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.

Spis treści

Part I. Isolation of Nuclei

 

1. Cell Type-Specific Affinity Purification of Nuclei for Chromatin Profiling in Whole Animals

            Florian A. Steiner and Steven Henikoff

           

2. Lysis Gradient Centrifugation – A Flexible Method for the Isolation of Nuclei from Primary Cells

            Karl Katholnig, Marko Poglitsch, Markus Hengstschläger, and Thomas Weichhart

 

3. Isolation of Nuclei in Media Containing an Inert Polymer to Mimic the Crowded Cytoplasm

            Ronald Hancock and Yasmina Hadj-Sahraoui

 

 

Part II. Nucleoli

 

4. A New Rapid Method for Isolating Nucleoli

            Zhou Fang  Li, Yun Wah Lam

 

5. Sequential Recovery of Macromolecular Components of the Nucleolus

            Baoyan Bai and Marikki Laiho

 

 

Part III. Genes and Chromatin

 

6. Au Nanoinjectors for Electrotriggered Gene Delivery into the Cell Nucleus

            Mijeong Kang and Bongsoo Kim

 

7. Improving Chromatin Immunoprecipitation (ChIP) by Suppression of Method-Induced DNA-Damage Signaling

            Sascha Beneke

 

8. Purification of Specific Chromatin Loci for Proteomic Analysis

Stephanie D. Byrum, Sean D. Taverna, and Alan J. Tackett

 

9. Chromatin Structure Analysis of Single Gene Molecules by Psoralen Crosslinking and Electron Microscopy

            Chris Brown, Julian Eskin, Stephan Hamperl, Joachim Griesenbeck, Melissa Jurica, Hinrich Boeger

 

10. Purification of Proteins on Newly Synthesized DNA Using iPOND

            Huzefa Dungrawala and David Cortez

 

11. Applying the Ribopuromycylation Method to Detect Nuclear Translation

            Alexandre David and Jonathan W. Yewdell

 

 

Part IV. The Intranuclear Milieu

 

12. Targeted Nano Analysis of Water and Ions in the Nucleus Using Cryo-Correlative Microscopy

            Frédérique Nolin, Dominique Ploton, Laurence Wortham, Pavel Tchelidze, Hélène Bobichon, Vincent Banchet, Nathalie Lalun, Christine Terryn, and Jean Michel

 

13. A Redox-Sensitive Yellow Fluorescent Protein Sensor for Monitoring Nuclear Glutathione Redox  Dynamics

Agata Banach-Latapy, Michèle Dardalhon, and Meng-Er Huang

 

 

Part V. Imaging Nuclear Structures

 

14. Determination of the Dissociation Constant of the NFkB p50/p65 Heterodimer in Living Cells Using Fluorescence Cross-Correlation Spectroscopy

Manisha Tiwari and Masataka Kinjo

 

15. Imaging and Quantification of Amyloid Fibrillation in the Cell Nucleus

            Florian Arnhold, Andrea Scharf, and Anna von Mikecz

 

16. Analysis of Nuclear Organization with TANGO, Software for High-Throughput Quantitative Analysis of 3D Fluorescence Microscopy Images

            Jean Ollion, Julien Cochennec. François Loll, Christophe Escudé, and Thomas Boudier

 

17. Quantitative Analysis of Chromosome Localization in the Nucleus

            Sandeep Chakraborty, Ishita Mehta, Mugdha Kulashreshtha, and B. J. Rao