Table Of ContentMethods in
Molecular Biology 1228
Ronald Hancock Editor
The Nucleus
Second Edition
M M B
ETHODS IN OLECULAR IOLOGY
Series Editor
John M. Walker
School of Life Sciences
University of Hertfordshire
Hat fi eld, Hertfordshire, AL10 9AB, UK
For further volumes:
http://www.springer.com/series/7651
The Nucleus
Second Edition
Edited by
Ronald Hancock
Laval University Cancer Research Centre-CRCHUQ Oncology, Québec, QC, Canada; Systems Biology Group,
Biotechnology Centre, Silesian University of Technology, Gliwice, Poland
Editor
Ronald Hancock
Laval University Cancer Research
Centre-CRCHUQ Oncology
Québec, QC, Canada
Systems Biology Group
Biotechnology Centre
Silesian University of Technology
Gliwice, Poland
ISSN 1064-3745 ISSN 1940-6029 (electronic)
ISBN 978-1-4939-1679-5 ISBN 978-1-4939-1680-1 (eBook)
DOI 10.1007/978-1-4939-1680-1
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Cover Image Caption: Water content in the nucleus of a HeLa cell; pixels containing 0 to 50 % water are yellow and
those containing 51 to 100 % water as a linear scale from light to dark blue (see Chapter 12).
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Prefa ce
This volume presents detailed recently developed protocols ranging from isolation of nuclei
to purifi cation 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 purifi cation of
cell type-specifi c 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 nanow-
ires is presented. The developing interest in analysis of specifi c regions of chromatin is
illustrated by protocols for the isolation and structural and proteomic analysis of chromatin
containing a single gene or containing newly synthesized DNA. A widely used method to
purify chromatin regions is immunoprecipitation (ChIP), but during isolation chromatin
structure may be modifi ed by DNA damage response systems, and conditions which allow
these artifacts to be avoided are described.
The concentration and localization of water and ions are crucial for macromolecular
interactions in the nucleus, and a new approach to measure these parameters by correlative
optical and cryo-electron microscopy is described. Similarly, redox conditions in the nucleus
have been little explored, and a method to follow the redox dynamics of nuclear glutathi-
one is an important step in this direction.
An important aspect of analyzing images of nuclear structures is the extraction of quan-
titative information, and this volume presents methods and software for high-throughput
quantitative analysis of 3D fl uorescence microscopy images, for quantifi cation of the forma-
tion of amyloid fi brils in the nucleus, and for quantitative analysis of chromosome territory
localization.
The friendly and timely collaboration of the contributors to this volume is greatly
appreciated.
Québec, QC, Canada Ronald Hancock
v
Contents
Preface. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v
Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . i x
PART I ISOLATION OF NUCLEI
1 Cell Type-Specific Affinity Purification of Nuclei for Chromatin
Profiling in Whole Animals. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Florian A . Steiner and S teven H enikoff
2 L ysis Gradient Centrifugation: A Flexible Method
for the Isolation of Nuclei from Primary Cells. . . . . . . . . . . . . . . . . . . . . . . . . 15
Karl Katholnig, Marko P oglitsch, M arkus H engstschläger,
and Thomas Weichhart
3 I solation of Nuclei in Media Containing an Inert Polymer
to Mimic the Crowded Cytoplasm . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 5
Ronald Hancock and Yasmina H adj-Sahraoui
PART II NUCLEOLI
4 A New Rapid Method for Isolating Nucleoli. . . . . . . . . . . . . . . . . . . . . . . . . . 3 5
Zhou F ang Li and Yun W ah Lam
5 S equential Recovery of Macromolecular Components of the Nucleolus. . . . . . 4 3
Baoyan B ai and Marikki L aiho
PART III GENES AND CHROMATIN
6 Au Nanoinjectors for Electrotriggered Gene Delivery
into the Cell Nucleus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 5
Mijeong K ang and B ongsoo K im
7 I mproving Chromatin Immunoprecipitation (ChIP)
by Suppression of Method-Induced DNA-Damage Signaling . . . . . . . . . . . . . 6 7
Sascha Beneke
8 P urification of Specific Chromatin Loci for Proteomic Analysis . . . . . . . . . . . . 8 3
Stephanie D. B yrum, S ean D . T averna, and Alan J . Tackett
9 C hromatin Structure Analysis of Single Gene Molecules
by Psoralen Cross-Linking and Electron Microscopy. . . . . . . . . . . . . . . . . . . . 93
Christopher R . Brown, Julian A . Eskin, S tephan Hamperl,
Joachim G riesenbeck, Melissa S. J urica, and Hinrich Boeger
10 Purification of Proteins on Newly Synthesized DNA Using iPOND . . . . . . . . 1 23
Huzefa Dungrawala and D avid C ortez
vii
viii Contents
11 Applying the Ribopuromycylation Method to Detect Nuclear Translation. . . . 1 33
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 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 45
Frédérique N olin, Dominique Ploton, Laurence Wortham,
Pavel T chelidze, Hélène B obichon, V incent B anchet,
Nathalie Lalun, Christine T erryn, and J ean Michel
13 A Redox-Sensitive Yellow Fluorescent Protein Sensor
for Monitoring Nuclear Glutathione Redox Dynamics. . . . . . . . . . . . . . . . . . . 159
Agata Banach-Latapy, M ichèle D ardalhon, and M eng-Er H uang
PART V IMAGING NUCLEAR STRUCTURES
14 Determination of the Dissociation Constant of the NFκB p50/p65
Heterodimer in Living Cells Using Fluorescence
Cross-Correlation Spectroscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 73
Manisha T iwari and Masataka K injo
15 I maging and Quantification of Amyloid Fibrillation in the Cell Nucleus . . . . . 1 87
Florian A rnhold, A ndrea S charf, and Anna v on Mikecz
16 Analysis of Nuclear Organization with TANGO,
Software for High-Throughput Quantitative Analysis
of 3D Fluorescence Microscopy Images. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 203
Jean O llion, Julien C ochennec, F rançois L oll, C hristophe Escudé,
and Thomas Boudier
17 Quantitative Analysis of Chromosome Localization in the Nucleus . . . . . . . . . 2 23
Sandeep C hakraborty, I shita M ehta, Mugdha Kulashreshtha,
and B. J . Rao
Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 35
Contributors
FLORIAN ARNHOLD • IUF – Leibniz Research Institute of Environmental Medicine at
Heinrich- Heine-University Duesseldorf , Duesseldorf, G ermany
BAOYAN BAI • Department of Radiation Oncology and Molecular Radiation Sciences,
The Johns Hopkins University School of Medicine , B altimore , M D , U SA ;
Sidney Kimmel Comprehensive Cancer Center, The Johns Hopkins University School
of Medicine , B altimore , MD, USA
AGATA BANACH-LATAPY • UMR3348 “Genotoxic Stress and Cancer,” Centre National de
la Recherche Scientifi que, Institut Curie, Orsay, France
VINCENT BANCHET • Laboratoire de recherche en Nanosciences EA 4682, UFR Sciences
Exactes et Naturelles , Université de Reims Champagne Ardenne , R eims, France
SASCHA BENEKE • Institute of Veterinary Pharmacology and Toxicology/Vetsuisse,
University of Zurich , Zurich, Switzerland
HÉLÈNE BOBICHON • CNRS UMR 7369, UFR Médecine, U niversité de Reims
Champagne Ardenne and CHU de Reims , Reims, France
HINRICH BOEGER • Department of Molecular, Cell and Developmental Biology,
University of California , Santa Cruz, C A , USA
THOMAS B OUDIER • Sorbonne Universités, U PMC Université Paris 06 , P aris , F rance
CHRISTOPHER R. BROWN • Department of Molecular, Cell and Developmental Biology,
University of California , Santa Cruz, C A , USA
STEPHANIE D. BYRUM • Department of Biochemistry and Molecular Biology,
University of Arkansas for Medical Sciences , Little Rock, A R , U SA
SANDEEP CHAKRABORTY • Department of Biological Sciences, T ata Institute
of Fundamental Research , M umbai, M aharashtra, I ndia ; P lant Sciences
Department, University of California , Davis, C A , USA
JULIEN C OCHENNEC • CNRS UMR7196, INSERM U565, Muséum National d’Histoire
Naturelle , Paris, France
DAVID C ORTEZ • Department of Biochemistry, V anderbilt University School of Medicine ,
Nashville, T N , U SA
MICHÈLE D ARDALHON • UMR3348 “Genotoxic Stress and Cancer,” Centre National de la
Recherche Scientifi que, Institut Curie , Orsay , F rance
ALEXANDRE DAVID • CNRS UMR-5203; INSERM U661; UM1; UM2, I nstitut de
Génomique Fonctionnelle , Montpellier, France
HUZEFA DUNGRAWALA • Department of Biochemistry , V anderbilt University School
of Medicine , Nashville, TN, USA
CHRISTOPHE ESCUDÉ • CNRS UMR7196, INSERM U565, Muséum National d’Histoire
Naturelle , P aris , France
JULIAN A. ESKIN • Department of Biology and Rosenstiel Basic Medical Science Research
Center , B randeis University , Waltham , M A , U SA
JOACHIM G RIESENBECK • Lehrstuhl fürBiochemie III, Biochemie-Zentrum Regensburg
(BZR), U niversität Regensburg , R egensburg, G ermany
ix
