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Methods in
Molecular Biology 1228

Ronald Hancock Editor

The Nucleus
Second Edition
METHODS IN MOLECULAR BIOLOGY

Series Editor
John M. Walker
School of Life Sciences
University of Hertfordshire
Hatfield, 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
Springer New York Heidelberg Dordrecht London

Library of Congress Control Number: 2014951833

© Springer Science+Business Media New York 2015

This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of the material is
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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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Preface

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 nanow-
ires is presented. The developing interest in analysis of specific 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 modified 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 fluorescence microscopy images, for quantification of the forma-
tion of amyloid fibrils 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 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix

PART I ISOLATION OF NUCLEI

1 Cell Type-Specific Affinity Purification of Nuclei for Chromatin
Profiling in Whole Animals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Florian A. Steiner and Steven Henikoff
2 Lysis Gradient Centrifugation: A Flexible Method
for the Isolation of Nuclei from Primary Cells . . . . . . . . . . . . . . . . . . . . . . . . . 15
Karl Katholnig, Marko Poglitsch, Markus Hengstschläger,
and Thomas Weichhart
3 Isolation of Nuclei in Media Containing an Inert Polymer
to Mimic the Crowded Cytoplasm . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
Ronald Hancock and Yasmina Hadj-Sahraoui

PART II NUCLEOLI
4 A New Rapid Method for Isolating Nucleoli . . . . . . . . . . . . . . . . . . . . . . . . . . 35
Zhou Fang Li and Yun Wah Lam
5 Sequential Recovery of Macromolecular Components of the Nucleolus . . . . . . 43
Baoyan Bai and Marikki Laiho

PART III GENES AND CHROMATIN

6 Au Nanoinjectors for Electrotriggered Gene Delivery
into the Cell Nucleus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55
Mijeong Kang and Bongsoo Kim
7 Improving Chromatin Immunoprecipitation (ChIP)
by Suppression of Method-Induced DNA-Damage Signaling . . . . . . . . . . . . . 67
Sascha Beneke
8 Purification of Specific Chromatin Loci for Proteomic Analysis . . . . . . . . . . . . 83
Stephanie D. Byrum, Sean D. Taverna, and Alan J. Tackett
9 Chromatin Structure Analysis of Single Gene Molecules
by Psoralen Cross-Linking and Electron Microscopy . . . . . . . . . . . . . . . . . . . . 93
Christopher R. Brown, Julian A. Eskin, Stephan Hamperl,
Joachim Griesenbeck, Melissa S. Jurica, and Hinrich Boeger
10 Purification of Proteins on Newly Synthesized DNA Using iPOND . . . . . . . . 123
Huzefa Dungrawala and David Cortez

vii
viii Contents

11 Applying the Ribopuromycylation Method to Detect Nuclear Translation . . . . 133

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 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 145
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. . . . . . . . . . . . . . . . . . . 159
Agata Banach-Latapy, Michèle Dardalhon, and Meng-Er Huang

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 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 173
Manisha Tiwari and Masataka Kinjo
15 Imaging and Quantification of Amyloid Fibrillation in the Cell Nucleus . . . . . 187
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. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 203
Jean Ollion, Julien Cochennec, François Loll, Christophe Escudé,
and Thomas Boudier
17 Quantitative Analysis of Chromosome Localization in the Nucleus . . . . . . . . . 223
Sandeep Chakraborty, Ishita Mehta, Mugdha Kulashreshtha,
and B. J. Rao

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 235
Contributors

FLORIAN ARNHOLD • IUF – Leibniz Research Institute of Environmental Medicine at

Heinrich- Heine-University Duesseldorf, Duesseldorf, Germany
BAOYAN BAI • Department of Radiation Oncology and Molecular Radiation Sciences,
The Johns Hopkins University School of Medicine, Baltimore, MD, USA;
Sidney Kimmel Comprehensive Cancer Center, The Johns Hopkins University School
of Medicine, Baltimore, MD, USA
AGATA BANACH-LATAPY • UMR3348 “Genotoxic Stress and Cancer,” Centre National de
la Recherche Scientifique, Institut Curie, Orsay, France
VINCENT BANCHET • Laboratoire de recherche en Nanosciences EA 4682, UFR Sciences
Exactes et Naturelles, Université de Reims Champagne Ardenne, Reims, France
SASCHA BENEKE • Institute of Veterinary Pharmacology and Toxicology/Vetsuisse,
University of Zurich, Zurich, Switzerland
HÉLÈNE BOBICHON • CNRS UMR 7369, UFR Médecine, Université de Reims
Champagne Ardenne and CHU de Reims, Reims, France
HINRICH BOEGER • Department of Molecular, Cell and Developmental Biology,
University of California, Santa Cruz, CA, USA
THOMAS BOUDIER • Sorbonne Universités, UPMC Université Paris 06, Paris, France
CHRISTOPHER R. BROWN • Department of Molecular, Cell and Developmental Biology,
University of California, Santa Cruz, CA, USA
STEPHANIE D. BYRUM • Department of Biochemistry and Molecular Biology,
University of Arkansas for Medical Sciences, Little Rock, AR, USA
SANDEEP CHAKRABORTY • Department of Biological Sciences, Tata Institute
of Fundamental Research, Mumbai, Maharashtra, India; Plant Sciences
Department,University of California, Davis, CA, USA
JULIEN COCHENNEC • CNRS UMR7196, INSERM U565, Muséum National d’Histoire
Naturelle, Paris, France
DAVID CORTEZ • Department of Biochemistry, Vanderbilt University School of Medicine,
Nashville, TN, USA
MICHÈLE DARDALHON • UMR3348 “Genotoxic Stress and Cancer,” Centre National de la
Recherche Scientifique, Institut Curie, Orsay, France
ALEXANDRE DAVID • CNRS UMR-5203; INSERM U661; UM1; UM2, Institut de
Génomique Fonctionnelle, Montpellier, France
HUZEFA DUNGRAWALA • Department of Biochemistry, Vanderbilt University School
of Medicine, Nashville, TN, USA
CHRISTOPHE ESCUDÉ • CNRS UMR7196, INSERM U565, Muséum National d’Histoire
Naturelle, Paris, France
JULIAN A. ESKIN • Department of Biology and Rosenstiel Basic Medical Science Research
Center, Brandeis University, Waltham, MA, USA
JOACHIM GRIESENBECK • Lehrstuhl fürBiochemie III, Biochemie-Zentrum Regensburg
(BZR), Universität Regensburg, Regensburg, Germany

ix
x Contributors

YASMINA HADJ-SAHRAOUI • Laval University Cancer Research Centre-CRCHUQ

Oncology, Québec, QC, Canada
STEPHAN HAMPERL • Lehrstuhl für Biochemie III, Biochemie-Zentrum Regensburg (BZR),
Universität Regensburg, Regensburg, Germany
RONALD HANCOCK • Laval University Cancer Research Centre-CRCHUQ Oncology,
Québec, QC, Canada; Systems Biology Group, Biotechnology Centre, Silesian University
of Technology, Gliwice, Poland
MARKUS HENGSTSCHLÄGER • Institute for Medical Genetics, Medical University of Vienna,
Vienna, Austria
STEVEN HENIKOFF • Basic Sciences Division, Howard Hughes Medical Institute, Seattle,
WA, USA; Fred Hutchinson Cancer Research Center, Seattle, WA, USA
MENG-ER HUANG • UMR3348 “Genotoxic Stress and Cancer,” Centre National de la
Recherche Scientifique, Institut Curie, Orsay, France
MELISSA S. JURICA • Department of Molecular, Cell and Developmental Biology, University
of California, Santa Cruz, CA, USA
MIJEONG KANG • Department of Chemistry, KAIST, Daejeon, South Korea
KARL KATHOLNIG • Institute for Medical Genetics, Medical University of Vienna,
Vienna, Austria
BONGSOO KIM • Department of Chemistry, KAIST, Daejeon, South Korea
MASATAKA KINJO • Laboratory of Molecular Cell Dynamics, Faculty of Advanced Life
Science, Hokkaido University, Sapporo, Japan
MUGDHA KULASHRESHTHA • Department of Biological Sciences, Tata Institute
of Fundamental Research, Mumbai, Maharashtra, India
MARIKKI LAIHO • Department of Radiation Oncology and Molecular Radiation Sciences,
The Johns Hopkins University School of Medicine, Baltimore, MD, USA
NATHALIE LALUN • CNRS UMR 7369, UFR Médecine, Université de Reims Champagne
Ardenne and CHU de Reims, Reims, Cedex, France
YUN WAH LAM • Department of Biology and Chemistry, City University of Hong Kong,
Kowloon, Hong Kong
ZHOU FANG LI • Department of Biology, South University of Science and Technology of
China, Shenzhen, Guangdong, P.R. China
FRANÇOIS LOLL • CNRS UMR7196, INSERM U565, Muséum National d’Histoire
Naturelle, Paris, France
ISHITA MEHTA • Department of Biological Sciences, Tata Institute of Fundamental
Research, Mumbai, Maharashtra, India; UM-DAE-Centre for Excellence
in Basic Sciences, Biological Sciences, Mumbai, Maharashtra, India
JEAN MICHEL • Laboratoire de Recherche en Nanosciences EA 4682, UFR Sciences Exactes
et Naturelles, Université de Reims Champagne Ardenne, Reims, France
ANNA VON MIKECZ • IUF – Leibniz Research Institute of Environmental Medicine at
Heinrich-Heine-University Duesseldorf Duesseldorf, Germany
FRÉDÉRIQUE NOLIN • Laboratoire de Recherche en Nanosciences EA 4682, UFR Sciences
Exactes et Naturelles, Université de Reims Champagne Ardenne, Reims, France
JEAN OLLION • CNRS UMR7196, INSERM U565, Muséum National d’Histoire
Naturelle, Paris, France
DOMINIQUE PLOTON • CNRS UMR 7369, UFR Médecine, Université de Reims
Champagne Ardenne and CHU de Reims, Reims, France
MARKO POGLITSCH • Attoquant Diagnostics GmbH, Vienna, Austria
 Contributors xi

B. J. RAO • Department of Biological Sciences, Tata Institute of Fundamental Research,

Mumbai, Maharashtra, India
ANDREA SCHARF • IUF – Leibniz Research Institute of Environmental Medicine at
Heinrich- Heine-University Duesseldorf, Duesseldorf, Germany
FLORIAN A. STEINER • Basic Sciences Division, Howard Hughes Medical Institute, Seattle,
WA, USA; Fred Hutchinson Cancer Research Center, Seattle, WA, USA
ALAN J. TACKETT • Department of Biochemistry and Molecular Biology,
University of Arkansas for Medical Sciences, Little Rock, AR, USA
SEAN D. TAVERNA • Department of Pharmacology and Molecular Sciences,
Johns Hopkins University School of Medicine, Baltimore, MD, USA
PAVEL TCHELIDZE • CNRS UMR 7369, UFR Médecine, Université de Reims Champagne
Ardenne and CHU de Reims, Reims, France
CHRISTINE TERRYN • Plate-forme IBISA, Université de Reims Champagne Ardenne,
Reims, France
MANISHA TIWARI • Laboratory for Nano-Bio Probes, Quantitative Biology Center, OLABB,
Osaka University, Suita, Japan
THOMAS WEICHHART • Institute for Medical Genetics, Medical University of Vienna,
Vienna, Austria
LAURENCE WORTHAM • Laboratoire de Recherche en Nanosciences EA 4682, UFR Sciences
Exactes et Naturelles, Université de Reims Champagne Ardenne, Reims, France
JONATHAN W. YEWDELL • Laboratory of Viral Diseases, National Institute of Allergy and
Infectious Diseases, Bethesda, MD, USA
 Part I

Isolation of Nuclei
 Chapter 1

Cell Type-Specific Affinity Purification of Nuclei

for Chromatin Profiling in Whole Animals
Florian A. Steiner and Steven Henikoff

Abstract
Analyzing cell differentiation during development in a complex organism requires the analysis of expression
and chromatin profiles in individual cell types. Our laboratory has developed a simple and generally
applicable strategy to purify specific cell types from whole organisms for simultaneous analysis of chromatin
and expression. The method, termed INTACT for Isolation of Nuclei TAgged in specific Cell Types,
depends on the expression of an affinity-tagged nuclear envelope protein in the cell type of interest. These
nuclei can be affinity-purified from the total pool of nuclei and used as a source for RNA and chromatin.
The method serves as a simple and scalable alternative to FACS sorting or laser capture microscopy to
circumvent the need for expensive equipment and specialized skills. This chapter provides detailed
protocols for the cell-type specific purification of nuclei from Caenorhabditis elegans.

Key words Nuclei, Cell type, Caenorhabditis elegans, Expression profiling, Chromatin profiling,
INTACT

1 Introduction

Most multicellular organisms are comprised of different tissues

and cell types. The differentiation of a specific cell type from undif-
ferentiated progenitors requires the expression of specific genes
at specific time points. This is achieved by a combination of
chromatin-based mechanisms involving transcription factor binding,
nucleosome remodeling, deposition of histone variants, and post-
translational histone modifications [1, 2]. As a consequence, each
cell type is characterized by a specific chromatin landscape that
gives rise to a specific gene expression signature. To understand cell
type-specific function and differentiation, it is important to under-
stand what chromatin changes underlie these processes and what
expression profiles arise from them. However, these questions
are intractable when studying whole organisms, as differences
in expression and chromatin profiles between cell types are

Ronald Hancock (ed.), The Nucleus, Methods in Molecular Biology, vol. 1228,
DOI 10.1007/978-1-4939-1680-1_1, © Springer Science+Business Media New York 2015

3
4 Florian A. Steiner and Steven Henikoff

marginalized in mixed populations. In recent years, several

approaches have been developed to obtain pure populations of
specific cell types or to extract RNA or DNA from specific cell types.
These methods include the use of cell lines derived from specific
tissues [3, 4], modified RNAs [5, 6], FACS-based approaches
either for dissociated cells or nuclei [7–12], Dam-methylase expres-
sion in specific tissues [13], laser capture microscopy [14–18], and
affinity-purification of nuclei [19–21].
Affinity-purification of nuclei is fast, simple and can be carried
out without specialized equipment. The method, termed isolation
of nuclei tagged in specific cell types (INTACT), relies on the
expression of an affinity tag on the nuclear envelope specifically in
the cell type of interest (Fig. 1).
The method was initially developed in our lab for Arabidopsis
thaliana and we have since adapted it to Caenorhabditis elegans
and Drosophila melanogaster [19–21]. Similar strategies have also
been developed by other labs to purify nuclei from D. melanogaster
and Xenopus [22, 23]. The method allows for the simultaneous
isolation of RNA and chromatin, thus allowing comparison of
gene expression profiles directly to the underlying chromatin
landscapes. We use a two-component system for nuclear tagging
where a nuclear pore fusion protein serves as a substrate for

Fig. 1 Scheme of tissue-specific nuclei purification. Nuclei are epitope-labeled

by the expression of a nuclear-tagging fusion protein specifically in the tissue of
interest. Total nuclei are released and mixed with magnetic beads which recognize
the epitope tag. The bead-bound nuclei are subsequently affinity-purified
 Cell Type-Specific Nuclei Purification 5

biotinylation by E. coli biotin ligase (BirA), which is co-expressed

in the same cells to mediate specific biotinylation. For C. elegans,
we selected the outer nuclear pore protein NPP-9 and fused it to a
tagging cassette that includes mCherry for visualization, biotin
ligase recognition peptide (BLRP), a preferred substrate for BirA,
and 3xFLAG for immunodetection. We call the NPP-9 fusion
protein nuclear tagging fusion (NTF). We express a BirA::GFP
fusion ubiquitously using the his-72 promoter to enable biotinyl-
ation of the NTF in vivo [21].
Application of the method to purify muscle nuclei from adult
C. elegans, using the myo-3 promoter to express tagged NPP-9 in
muscle, resulted in yields of 1–2 million nuclei with >90 % purity.
Analysis of these nuclei revealed hundreds of genes that were
specifically upregulated in muscle tissue, but also showed that the
nucleosome occupancy was reduced over the promoters and
within the bodies of these genes. The method also greatly increased
the sensitivity of detecting changes in gene expression upon
knock-down of the muscle-specific transcription factor HLH-1,
underlining the importance of analyzing pure populations of nuclei
when analyzing changes that only affect a subset of cells within an
organism [21].
Here we provide a detailed protocol for the purification of
tissue-specific nuclei from C. elegans. We divide the protocol into
three stages: (1) C. elegans culture and fixation; (2) Isolation
and affinity-purification of nuclei; and (3) Assessment of quality.
The purified nuclei can be used as a source of RNA for gene expres-
sion profiling, of chromatin for micrococcal nuclease- or sonication-
mediated fragmentation and chromatin immunoprecipitation, or
of proteins for proteomic profiling.

2 Materials

2.1 Culture 1. Peptone-rich agar plates, 150 mm diameter: 12.5 g agar, 10 g

and Fixation peptone, 0.6 g NaCl, 1.5 g KH2PO4, 0.25 g K2HPO4, H2O
of C. elegans Adults to 1 L.
2. E. coli strain NA22. Available from the Caenorhabditis
Genetics Center (CGC), http://www.cbs.umn.edu/research/
resources/cgc.
3. C. elegans strain expressing both NTF and BirA, e.g., strain
JJ2300 expressing NTF in body wall muscle cells and BirA
ubiquitously, available from the CGC.
4. C. elegans strain expressing only NTF (negative control), e.g.,
strain JJ2286 expressing NTF in body wall muscle cells,
available from the CGC.
5. M9 solution: 22 mM KH2PO4, 42 mM Na2HPO4, 86 mM
NaCl, 1 mM MgSO4.
6 Florian A. Steiner and Steven Henikoff

6. Phosphate-buffered saline (PBS): 8 mM Na2HPO4, 1.46 mM

KH2PO4, 137 mM NaCl, 2.7 mM KCl, pH 7.4.
7. 50-mL conical tubes.
8. Sodium hypochlorite solution, 10–15 % available chlorine
(Sigma).
9. 5 M NaOH.
10. N,N-dimethylformamide (Sigma).

2.2 Isolation and 1. Nuclei purification buffer (NPB): 10 mM Tris pH 7.5, 40 mM

Affinity-Purification NaCl, 90 mM KCl, 2 mM ethylenediaminetetraacetic acid
of Nuclei (EDTA), 0.5 mM ethylene glycol-bis(2-aminoethylethes)-
N1N1N;N1– tetraacetic acid (EGTA), 0.5 mM spermidine,
0.2 mM spermine, 0.2 mM DTT, 0.1 % Triton X-100.
2. NPB-0.5: NPB supplemented with Triton X-100 (0.5 % v/v).
3. Ceramic mortar and pestle.
4. Liquid nitrogen.
5. Glass dounce homogenizer, 7 mL.
6. Refrigerated tabletop centrifuge.
7. Bioruptor sonicator (Diagenode).
8. Refrigerated centrifuge for 50-mL conical tubes with swinging-
bucket rotor (e.g., Sorvall Instruments RC5C).
9. Streptavidin M-280 Dynabeads (Invitrogen).
10. End-over-end rotator for Eppendorf tubes (e.g., Labquake
Shaker, Thermo Scientific).
11. MiniMacs magnet (Miltenyi Biotec).
12. 10-mL serological pipettes.
13. NPB supplemented with 1 % (w/v) bovine serum albumin
(BSA).
14. 1-mL micropipette tips.
15. Tygon tubing.
16. Hoffman tubing clamp.

2.3 Quality Control 1. 4′,6-diamidino-2-phenylindole (DAPI).

and Estimation 2. Glass coverslips, 22 × 40 mm, No 1.5.
of Yield
3. Microscope slides 3 well, 25 × 75 mm.
4. Upright fluorescence microscope (e.g., Zeiss Axioplan).
5. SDS-PAGE gel loading buffer (2×): 10 mM Tris–HCl,
pH 6.8, 4 % (w/v) SDS, 20 % (v/v) glycerol, 2 % (v/v)
β-mercaptoethanol, 0.04 % (w/v) bromophenol blue.
6. 100 °C heat block.
7. 6 and 18 % Tris–Glycine polyacrylamide gels.
 Cell Type-Specific Nuclei Purification 7

8. Western blot transfer system.

9. Nitrocellulose membranes.
10. 2 % (w/v) bovine serum albumin (BSA) in PBS.
11. Anti-histone H3 C-terminus antibody (Abcam).
12. Anti-FLAG M2 antibody (Sigma).
13. Horseradish peroxidase (HRP)-conjugated streptavidin.
14. HRP-conjugated anti-rabbit and anti-mouse secondary
antibodies.
15. Autoradiography film (Kodak).

3 Methods

3.1 Culture and 1. Seed peptone-rich plates with 2 mL of an overnight culture of

Fixation of Adult E. coli NA22. Incubate the plates overnight at 37 °C, then
C. elegans overnight at room temperature.
2. Grow the worm strain expressing both NTF and BirA (sample)
and the strain expressing only NTF (negative control) on these
plates until they are almost starved (see Note 1).
3. Wash the worms off the plates with M9 solution.
4. Wash the worms three times in M9 and resuspend them in
10 mL of M9 in a 50-mL conical tube.
5. Add 1 mL of sodium hypochlorite solution and 1 mL of 5 M
NaOH.
6. Incubate with occasional shaking until the worm bodies have
mostly disappeared, leaving behind embryos.
7. Pellet the embryos by centrifugation at 2,000 × g for 1 min.
8. Wash the embryos three times with M9.
9. Plate the embryos on fresh NA22 plates (see Note 2).
10. Grow synchronized cultures to the young adult stage.
11. For one preparation, use worms from 2 to 4 plates or about
1–2 mL worm pellet, 1.5–2 million worms (see Note 3).
12. Wash the worms off the plates with M9 and collect in 50-mL
conical tubes.
13. Pellet the worms to the bottom of the tubes by incubation on
ice for 10–15 min.
14. Wash the worms 3–4 times with M9 and twice with PBS.
15. Centrifuge at 1,000 × g for 2 min, remove residual PBS.
16. Add N,N-dimethylformamide cooled to −20 °C to a volume
of 50 mL (see Notes 4 and 5).
17. Incubate at room temperature for 1 min (see Note 6).
8 Florian A. Steiner and Steven Henikoff

18. Pellet the worms by centrifugation at 1,000 × g for 1 min.

19. Remove the N,N-dimethylformamide and wash the worms
three times with PBS cooled at 4 °C.
20. Freeze the worms dropwise in liquid nitrogen (see Note 7).
21. Grind the worms to a fine powder under liquid nitrogen.

3.2 Isolation and 1. All subsequent steps are carried out on ice or in the cold room.
Affinity-Purification Buffers and equipment are precooled.
of Nuclei 2. Add NPB to the worm powder to a total volume of 6 mL.
3. Transfer the suspension to a glass dounce homogenizer.
4. Break up the tissues with 30 strokes of the tight-fitting piston.
5. Distribute into six 1.5-mL centrifuge tubes.
6. Pellet debris by centrifugation at 100 × g for 2 min in a refrig-
erated tabletop centrifuge.
7. Collect the supernatants containing nuclei and pool in a
50-mL conical tube.
8. Resuspend each pellet in 1 mL of NPB.
9. Sonicate with a Bioruptor sonicator at the lowest power
output setting (130 W) twice for 30 s to release more nuclei
(see Notes 8 and 9).
10. Pellet debris by centrifugation at 100 × g for 2 min in a refrig-
erated tabletop centrifuge.
11. Collect the supernatants containing nuclei, and pool in the
same 50-mL conical tube as above.
12. Repeat the sonication, low speed centrifugation and supernatant
collection two more times.
13. Discard the pellets, which contain worm fragments and debris
(see Note 10).
14. Bring the volume of nuclei collected in the 50-mL conical
tube to 50 mL with NPB.
15. Pellet residual debris by centrifugation at 100 × g for 5 min in
a refrigerated centrifuge, then transfer the supernatant to a
new 50-mL conical tube, discarding the pellet.
16. Add a 3-mL cushion of OptiPrep at the bottom of the tube
(see Note 11).
17. Collect the nuclei as a layer on the cushion by centrifugation
at 1,000 × g for 10 min in a refrigerated centrifuge (see Note 12).
18. Transfer the nuclei into new 50-mL tube (see Note 13).
19. Bring the volume to 50 mL with NPB, add an OptiPrep cush-
ion, and collect the nuclei on the cushion by centrifugation at
1,000 × g for 10 min in a refrigerated centrifuge. Repeat for a
total of three centrifugation steps (see Note 14).
 Cell Type-Specific Nuclei Purification 9

20. Collect the nuclei in a 15-mL conical tube.

21. Retain 5 % of the sample for Western blot analysis and 1 % for
microscopy (see Subheading 3.3, steps 1 and 6).
22. Bring the volume to 5 mL with NPB.
23. Add 30 μL of washed magnetic streptavidin-coated Dynabeads
(see Notes 15 and 16).
24. Incubate for 45 min at 4 °C using an end-over-end rotator.
25. Bring the volume to 10 mL with NPB-0.5 (final concentration
of Triton X-100 is 0.3 %).
26. Incubate 1-mL micropipette tips in NPB supplemented with
1 % (w/v) BSA (see Note 17).
27. Draw the nuclei into a 10-mL serological pipette.
28. Insert the pipette into a 1-mL micropipette tip that is wedged
into a MiniMacs magnet. A schematic view of the assembled
column is shown in Fig. 2.
29. Run the nuclei through the column at a flow rate of 1 mL/
min. Control the flow rate with a Hoffman tubing clamp.

Fig. 2 Column setup for affinity purification. A 10-mL serological pipette is

inserted into a 1-mL pipette tip which has been wedged into a MiniMacs magnet.
A piece of Tygon tubing is attached to the pipette tip, and flow is controlled by a
Hoffmann tubing clamp
10 Florian A. Steiner and Steven Henikoff

30. Elute the beads into 10 mL of NPB-0.5.

31. Run the eluate through a second column (see Subheading 3.2,
steps 26–30 and Note 18).
32. Elute the beads into 500 μL of NPB.
33. Retain 5 % of the eluate for Western blot analysis and 1 % for
microscopy (see Subheading 3.3, steps 1 and 6).
34. The remaining nuclei can be used for RNA, chromatin or pro-
tein isolation.

3.3 Quality Control 1. To 1 % aliquots collected at steps 21 and 33 of Subheading 3.2,

and Estimation add DAPI to a final concentration of 1 μg/mL.
of Yield 2. Place a 1-μL aliquot of this sample onto a 3-well microscope
slide.
3. Cover with a coverslip.
4. Count bead-bound vs. non-bead-bound nuclei under a com-
pound microscope (see Note 19).
5. Compare to the number of nuclei in the negative control.
6. Mix 5 % aliquots collected at steps 21 and 33 of Subheading 3.2
with equal amounts of SDS-PAGE sample buffer.
7. Incubate at 100 °C for 5–10 min.
8. Resolve the samples on two 6 % SDS-PAGE gels (one for
detection of the FLAG epitope and one for detection of the
biotinylated BLRP) and on one 18 % gel (for detection of his-
tone H3).
9. Transfer the proteins to nitrocellulose membranes using a
Western blot transfer system.
10. Block the membranes with 2 % (w/v) BSA in PBS (see Note
20).
11. Detect biotinylated BLRP with streptavidin-HRP, FLAG with
anti-FLAG antibody followed by anti-mouse-HRP antibody,
and histone H3 with anti-H3 antibody followed by anti-
rabbit-HRP antibody (see Note 21).
12. Expose the membranes to autoradiography film.

4 Notes

1. We use a negative control for every purification to assess the

quality of the pull-down. Nuclear lysis leads to clumping of
the nuclei that can subsequently stick to the magnetic beads
and lead to a large number of false positive nuclei. This prob-
lem is most easily recognized when a negative control is used.
The problem of false positives caused by nuclear lysis and
clumping is best addressed by gentler handling of the nuclei.
 Cell Type-Specific Nuclei Purification 11

2. Treating adult worms with sodium hypochlorite and letting

embryos hatch on fresh NA22 plates generates age-synchronized
populations. This is important, as cells from the same tissue
will have different expression and chromatin profiles depend-
ing on the stage of development.
3. The amount of worms needed to prepare nuclei of a given cell
type will vary greatly depending on the required yield, the
abundance of the cell type, and the efficiency of release of
these nuclei from surrounding tissue. The first and third
factors need to be determined empirically.
4. The purification can be done without fixation, as we have
demonstrated for D. melanogaster mesoderm nuclei [21]. For
native purifications, we omit detergents from the NPB to pre-
vent lysis of the nuclei and use HB125 buffer (0.125 M
sucrose, 15 mM Tris, pH 7.5, 15 mM NaCl, 40 mM KCl,
2 mM EDTA, 0.5 mM EGTA, 0.5 mM spermidine, 0.15 mM
spermine, Roche Complete protease inhibitor cocktail) instead
of NPB. However, when using the C. elegans NPP-9 tag we
found that avoidance of any fixative leads to lower yields, pos-
sibly due to dissociation of NPP-9 from the nuclear pore.
5. 1 % formaldehyde can be used in the place of DMF for
fixation. In this case, the 10 mM Tris is replaced by 50 mM
HEPES in the NPB buffer because Tris quenches formalde-
hyde. We recommend the use of formaldehyde cross-linking
if chromatin immunoprecipitation is the downstream
application.
6. It is important to fix the worms lightly, as over-fixing will
hinder the liberation of nuclei from the surrounding tissue.
7. Worms can be kept at −80 °C for a few days. However, we
have experienced a reduction in pull-down efficiency upon
longer storage.
8. The sonication steps may be unnecessary for some tissues,
e.g., germ cells.
9. It is important to sonicate lightly. Too much sonication
will lead to the formation of small debris that can stick to
nuclei and beads and will decrease the purity of the final
preparation.
10. The pellet can be examined under a microscope for the degree
of fragmentation and presence of labeled nuclei.
11. We use an OptiPrep cushion to prevent nuclei from being
forced against the wall of the conical tube, which would cause
lysis and clumping.
12. It is important to use a swinging bucket rotor so that the
nuclei are pelleted on top of the OptiPrep cushion and do not
collect on the side of the tube.
12 Florian A. Steiner and Steven Henikoff

13. We collect the nuclei by first removing the supernatant,

then removing the OptiPrep through the layer of nuclei,
to leave the nuclei in approximately 500–1,000 μL in the
conical tube.
14. Washing the nuclei is necessary because of the abundance of
endogenous biotinylated proteins in C. elegans, which
compete with the biotinylated BLRP tag for binding of the
streptavidin beads. As most of these proteins are cytoplasmic,
background levels can be reduced by washing the nuclei.
We found that a minimum of two washes are necessary.
15. It is possible to use beads conjugated to an anti-FLAG
antibody to affinity-purify the nuclei via FLAG-tag without
the need for in vivo biotinylation by BirA. We found that this
reduced the purity of the affinity-purified nuclei from >90 to
80–90 %.
16. We tested several different sizes of beads and found that 2-μm
beads gave the best results because of their size relative to the
nuclei. Larger beads crushed the nuclei, whereas smaller beads
were less efficient in capturing the nuclei.
17. Coating the 1-mL tip with BSA reduces sticking of the beads
and nuclei to the tip, which increases the yield. BSA from
some sources can contain RNases and should be avoided when
the purified nuclei are used for expression profiling.
18. We found that two passes over the column give the best com-
bination of yield and purity.
19. Bead-bound nuclei are considered positives, and non-bead-
bound nuclei are considered false positives. The ratio of the
number of non-bead-bound to bead-bound nuclei represents
the purity of the pull-down, which should be >0.9. The purity
can also be assessed by counting mCherry-positive and
mCherry-negative nuclei. However, this approach is less prac-
tical, as there is strong auto-fluorescence of the magnetic
beads. We have found that the two approaches result in very
similar numbers. From the number of bead-bound nuclei and
the size of the aliquot, the total number of nuclei in the pull-
down sample (yield) can be extrapolated.
20. Milk should be avoided as a blocking agent, as it contains
biotin that will cause background when probing with
streptavidin-HRP.
21. Streptavidin detection tests for the successful in vivo biotinyl-
ation of the BLRP tag within the NTF. However, the signal is
often relatively weak, possibly due to the presence of relatively
large amounts of endogenous biotinylated proteins. We there-
fore also routinely detect the NTF with an anti-FLAG antibody,
which also confirms the size of the NTF. We detect histone
 Cell Type-Specific Nuclei Purification 13

Fig. 3 Representative Western blots. Blots were probed by anti-FLAG, streptavi-

din, and anti-histone H3. The FLAG tag (top) is present in both input samples, but
only in the successful pull-down, indicating the successful pull-down of the
nuclear-tagging fusion (NTF) protein. Streptavidin (middle) confirms the in vivo
biotinylation of the NTF. Endogenous biotinylated proteins are also visible and
marked with an asterisk. Histone H3 (bottom) is only present in the successful
pull-down, confirming that nuclei and not just the NTF were pulled down (image
reproduced from [21])

H3 in the pull-down samples to confirm successful purification

containing chromatin. Detection of histone H3 is also very
sensitive to false positives in the negative control. Representative
Western blots are shown in Fig. 3.

Acknowledgements

This work was supported by HHMI, NIH (U01-HG004274),

and the Swiss National Science Foundation (PBSKP3-124362).

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 Chapter 2

Lysis Gradient Centrifugation: A Flexible Method

for the Isolation of Nuclei from Primary Cells
Karl Katholnig, Marko Poglitsch, Markus Hengstschläger,
and Thomas Weichhart

Abstract
The isolation of nuclei from eukaryotic cells is essential for studying the composition and the dynamic
changes of the nuclear proteome to gain insight into the mechanisms of gene expression and cell signal-
ling. Primary cells are particularly challenging for standard nuclear isolation protocols due to low protein
content, sample degradation, or nuclear clumping. Here, we describe a rapid and flexible protocol for the
isolation of clean and intact nuclei, which results in the recovery of 90–95 % highly pure nuclei. The
method, called lysis gradient centrifugation (LGC), is based on an iso-osmolar discontinuous iodixanol-
based density gradient including a detergent-containing lysis layer. A single low g-force centrifugation step
enables mild cell lysis and prevents extensive contact of the nuclei with the cytoplasmic environment. This
fast method shows high reproducibility due to the relatively little cell manipulation required by the inves-
tigator. Further advantages are the low amount of starting material required, easy parallel processing of
multiple samples, and isolation of nuclei and cytoplasm at the same time from the same sample.

Key words Nuclear isolation, Mild lysis, Fractionation, Single step, Discontinuous density gradient

1 Introduction

The spatial separation of the nuclear and the cytoplasmic compart-

ment plays an essential role in the regulation of gene expression in
all immune cells [1]. Gaining information about the composition
and the dynamic changes in the nuclear proteome is important for
understanding the mechanisms of gene regulation and cell signal-
ing [2–4]. A number of protocols aim to isolate pure and intact
nuclei from eukaryotic cells for further analysis by western blots,
electrophoretic mobility shift assays (EMSA), or mass spectrome-
try [3–8]. The most important and initial step of frequently
employed nuclear isolation protocols is the disruption of the cyto-
plasmic membrane, while the nuclear membrane should remain
intact. This is usually achieved by induction of cell swelling through

Ronald Hancock (ed.), The Nucleus, Methods in Molecular Biology, vol. 1228,
DOI 10.1007/978-1-4939-1680-1_2, © Springer Science+Business Media New York 2015

15
16 Karl Katholnig et al.

the incubation of the cells in a hypotonic buffer for a tightly con-

trolled period of time followed by mechanical or chemical disruption
of the cell membrane [9]. NP-40 is a nonionic non-denaturing
detergent and was for a long time the most frequent detergent
used in nuclear isolation protocols. It was either directly included
into the hypotonic lysis buffer or added after cell swelling [10, 11].
As NP-40 is no longer commercially available, it was replaced by
the chemically indistinguishable IGEPAL CA-630. Mechanical
stress is often used to aid plasma membrane disruption and is intro-
duced either by defined vortexing times or by passing the cells
through a syringe. The crude lysates containing the nuclei are then
usually washed by pelleting the nuclei by centrifugation.
Alternatively, Graham and coworkers developed a method to purify
the nuclei from the crude lysate based on a discontinuous density
gradient formed by iodixanol followed by centrifugation and
nuclear banding [9]. This method has been used frequently to
study nuclear proteins [12–14].
From a biochemical point of view, the purification of nuclei
from mammalian cells is associated with many technical problems
[11]. Temperature control cannot be assured easily at any time of
the nuclear isolation procedure during mechanical disruption
methods, and moreover the nuclei are in extensive contact with the
cytoplasmic environment containing lysis-activated proteases that
lead to partial degradation and modification of the samples despite
the presence of protease inhibitors [15, 16]. Repeated washing
steps, necessary to remove nucleus-associated membrane systems
and cytoskeletal components, may result in nuclear leakage due to
overexposure to detergent and mechanical stress. Therefore,
nuclear leakage results not only in loss of target proteins but also in
clumping of the nuclei due to released chromosomal DNA [17].
Clumping leads to a low recovery of nuclei, explaining the large
amount of starting material required by standard fractionation
procedures. Many protocols are optimized for distinct cell types as
the nuclear density and stability show profound variations among
cell types and stimulation procedures [18, 19]. All of these caveats
are true for cell lines and primary cells; however, it is particularly
difficult to get reliable and clean nuclear preparations from primary
cells which are available only in a limited amount and have low
protein content [20].

2 Materials

2.1 Cells This method has been used successfully for:

Primary cells: CD14+ monocytes; macrophages (MΦ); T
lymphocytes.
Cell lines: JE-6 (Jurkat cells, an immortalized line of T lympho-
cytes); RAJI (a lymphoblastoid cell line derived from a Burkitt
 Isolation of Nuclei by a Single Centrifugation Step 17

lymphoma); MEF (murine embryonic fibroblasts); THP-1

cells (a promonocytic cell line derived from a human acute
lymphocytic leukemia patient) (see Note 1).

2.2 Lysis Gradient 1. Visipaque™ 320 (65.2 % Iodixanol; GE Healthcare).

Preparation 2. Phosphate-buffered saline solution (PBS): 210.0 mg/L
KH2PO4, 9,000 mg/L NaCl, 726.0 mg/L Na2HPO4-7H2O
(Lonza).
3. Protease inhibitor cocktail: 10× solution made by dissolving
one tablet of cOmplete EDTA-free (Roche) in 2 mL of PBS.
4. Coomassie Brilliant Blue R250 (Bio-Rad).
5. Crystal Violet (Sigma-Aldrich).
6. IGEPAL CA-630 (Sigma).
7. Polystyrene tubes: 8 mL, 13 × 100 mm (BD Biosciences).
8. Crushed ice.

2.3 Nuclear Gradient 1. Standard refrigerated laboratory centrifuge (e.g., Rotanta

Centrifugation 460RS, Hettich) equipped with a swinging bucket rotor.
2. Vacuum-attached glass pipette.

2.4 Western Blots 1. Denaturing SDS-sample buffer (4×): 250 mM Tris–HCl,

pH 6.8, 40 % glycerol, 8 % SDS, 400 mM dithiothreitol, and
0.04 % (w/v) bromophenol blue.
2. Nitrocellulose membranes: Protran, Whatman.
3. PBS-T: 0.05 % Tween-20 in PBS.
4. Blocking solution: 4 % dry milk (Bio-Rad) in PBS-T.
5. Primary antibodies: Rabbit anti-GAPDH mAb (Cell Signaling)
1:1,000 in PBS-T + 0.02 % NaN3; rabbit anti-ABTF-IID poly-
clonal (Santa Cruz) 1:500 in PBS-T + 0.02 % NaN3.
6. Secondary antibody: ECL donkey anti-rabbit IgG, HRP-linked
whole Ab [GE Healthcare): 1:3,000 in PBS-T + 4 % dry milk.
7. HRP Substrate: Immobilon Western Chemiluminescent
(Millipore).
8. Chemiluminescence imager.

3 Methods

Our method is based on an iodixanol-based gradient [21] where

we included a lysis layer containing the detergent IGEPAL CA-630
[22]. The incorporation of a lysis layer allows the isolation of intact
nuclei from living cells in a single centrifugation step, and therefore
minimizes the exposure of the nuclei to cytoplasmic protease activity.
The gradient for LGC is built in such a way that the whole suspen-
sion of living cells can be directly loaded onto the iodixanol-based
18 Karl Katholnig et al.

Fig. 1 (a) Principle of the method. The living cells are applied on top of the gradient in the original culture
medium. During 10 min of low gravity centrifugation in a swinging bucket rotor, the cells pass through different
functional layers including the cell wash layer (CW) and the lysis layer (L), at the beginning of which the cyto-
plasmic membrane is disrupted. Cytoplasmic proteins (C) remain on top of the lysis layer. Nuclei continue to
pass through the nuclei wash layer (NW) and form a band on top of the floating layer (N). (b) A nuclear gradient
purification performed with 2 × 106 THP-1 cells. The cells were centrifuged for the indicated times at 1,000 × g

gradient in its original culture medium (Fig. 1). The discontinuous

iso-osmolar lysis gradient allows a g-force driven discrimination
between unlysed and lysed cells and clean and ER-contaminated
nuclei during centrifugation. A band of pure nuclei accumulates at
the lowest interface, while nuclei contaminated with other cellular
structures are trapped at upper layers due to their lower density (see
Note 2). The cells can be stained with Crystal Violet and Coomassie
Blue in order to visualize the bands occurring in the gradient and
therefore enables easy harvesting of the nuclei.

3.1 Preparation 1. Prepare iodixanol dilutions up to 40 % (w/v) by diluting

of the Lysis Gradient Visipaque 320 in PBS (see Note 3). The volume of Visipaque
and PBS for the different layers of the gradient can be found in
Table 1. Add one-tenth volume of 10× protease inhibitor solu-
tion to the cell lysis layer and nuclei wash layer immediately
before use.
2. Add Coomassie Brilliant Blue to the lysis layer to a final con-
centration of 4 μg/mL (see Note 4).
3. Add Crystal Violet to the cell suspension and lysis layer to a
final concentration of 5 μg/mL (see Note 4).
4. Add IGEPAL CA-630 to the lysis layer to a final concentration
of 0.5 % (v/v).
5. Prepare lysis gradients in 8 mL polystyrene tubes suitable for
2 × 105–5 × 106 cells. The gradients are set up by sequentially
overlaying 1 mL of floating layer with 0.5 mL of nuclei wash
 Isolation of Nuclei by a Single Centrifugation Step 19

Table 1
Composition of LGC gradients for isolating nuclei from different cell types

Primary cells Cell lines

Iodixanol Density PBS Visipaque
[% (w/v)] [g/cm3] [mL] [mL] CD14+ MΦ T-Cells THP-1 JE-6 RAJI MEF
0 1.016 10.0 0.0
a
5 1.051 9.2 0.8 CW CW CW CW CW CW CW
10 1.071 8.5a 1.5 L L L L L L L
15 1.100 7.7 2.3
20 1.129 6.9 3.1 NW NW
25 1.155 6.2 3.8 NW NW NW NW NW
30 1.178 5.4 4.6
35 1.199 4.6 5.4 F F F F F F F
40 1.234 3.9 6.1
65.2 1.372 0.0 10.0
CW cell wash layer, L lysis layer, NW nucleus wash layer, F floating layer
a
Total volume; includes 1 mL of 10× protease inhibitor solution added immediately before use

layer, 1 mL of lysis layer, and 0.5 mL of cell wash layer. All

these steps are carried out on ice (see Note 5).
6. Stain the cooled cell suspension with 5 μg/mL Crystal Violet
(see Note 4).
7. Apply the cooled cell suspension in up to 1 mL of the
original culture medium immediately before lysis gradient
centrifugation.

3.2 Nuclear Gradient 1. After setting up the lysis gradient and adding the cell suspen-
Centrifugation sion, centrifuge at 1,000 × g for 10 min at 4 °C in a refrigerated
and Harvesting centrifuge equipped with a swinging bucket rotor.
of Fractions 2. After centrifugation, place the tubes carefully on ice.
3. Gently remove the culture medium as well as the upper part of
the cell wash layer using a vacuum-attached glass pipette.
4. The upper 500 μL of the lysis layer are collected with a 1 mL
Gilson pipette for the analysis of cytoplasmic fractions.
5. The residual lysis layer and the upper half of the nuclei wash
layer are removed with a vacuum-attached glass pipette.
6. Collect the light blue band of nuclei (150 μL) between the
nuclei wash layer and the floating layer (see Note 6).
20 Karl Katholnig et al.

7. For western blot analysis, lyse the nuclei directly by adding

50 μL of 4× reducing SDS-PAGE sample buffer followed by
denaturation for 5 min at 95 °C (see Note 7).

3.3 Western Blots 1. Lyse the harvested fractions by adding one volume of 4×
reducing SDS-PAGE sample buffer to three volumes of the
fractions.
2. Denature proteins at 95 °C for 5 min.
3. Cool the samples to 10 °C and centrifuge in a microcentrifuge
for 2 min at 25,000 × g.
4. Subject the supernatants to SDS-PAGE and blot the gels onto
nitrocellulose membranes.
5. Incubate the membranes in blocking solution for 1 h at room
temperature.
6. Incubate the membranes with primary antibodies at 4 °C
overnight. In order to confirm the purity of the nuclear and
cytoplasmic fractions, make use of antibodies against GAPDH
and TF-IID: GAPDH should only be seen in the cytoplasmic
fractions and the transcription factor TF-IID should only be
present in the nuclear fraction (Fig. 2).
7. Wash the membranes 3× with PBS-T and add the horserad-
ish peroxidase-labeled secondary antibody for 1 h at room
temperature.

Fig. 2 Purity and integrity of LGC-isolated nuclei. (a) 106 THP-1 cells were
subjected to LGC followed by taking aliquots of proteins for western blots from
the cytoplasmic fraction (C), the lower interface of lysis layer (1 ), the central
nuclear washing layer (2 ), and the nuclear band (N). Equal volumes of the indicated
layers were western blotted and immunolabeled for Calnexin, GAPDH, and TF-IID.
(b) Aliquots of nuclear (NUC) and cytoplasmic (CYT) fractions obtained by LGC
from 106 THP-1 cells per treatment condition were investigated by western
blotting for GAPDH and TF-IID. Total lysates (TL) in the same treatment conditions
are shown as control
 Isolation of Nuclei by a Single Centrifugation Step 21

8. Wash the membranes 3× with PBS-T and detect antibody-

labeled proteins with Immobilon Western Chemiluminescent
HRP Substrate.
9. Detect the signals using a chemiluminescence imager.

4 Notes

1. This method can be used for the isolation of nuclei from virtu-
ally any cell type by just optimizing the densities of the differ-
ent functional layers. It is also suitable for isolating nuclei from
only a limited amount of cells; for example we have isolated
nuclei from 5 × 105 primary macrophages and successfully per-
formed western blotting. After an initial trypsinization step,
adherent cells can also be processed with this protocol.
2. In principle, LGC can be adapted for ultracentrifugation to
isolate cell organelles such as mitochondria, lysosomes, or per-
oxisomes requiring only a layer with the correct density to be
present in the gradient [23, 24].
3. Visipaque 320 containing iodixanol is widely used as an intra-
venous contrast agent in clinical radiology, and the amount
discarded from single-use containers can reach up to 50 % of
the pack volume. The remaining Visipaque 320 provides a
cheap and easily available source of iodixanol with the major
advantage of iso-osmolarity, in contrast to OptiPrep which is
a 60 % aqueous solution of Iodixanol whose iso-osmolarity
after dilution cannot be controlled as precisely as with
Visipaque [11].
4. If you want to carry out more sensitive methods than western
blot after the LGC, it is recommended to avoid the use of
Coomassie Brilliant Blue and Crystal Violet. The gradient can
also be carried out without the addition of these dyes, which
might interfere with more sensitive assays.
5. To minimize mixture of the layers when preparing the lysis
gradient, the solutions should flow down the tube wall slowly
and tubes should be placed on ice before the addition of the
next layer. By omitting the lowest floating layer, the nuclei can
be easily pelleted although this might influence their purity
due to proteins sticking to the upper portion of the tubes.
6. Do not penetrate the wash layers with the pipette in order to
harvest the nuclei, as proteins from the wash layer might stick
to the pipette tip and contaminate the nuclei. It is highly rec-
ommended to suck off the medium and the upper half of the
cell wash layer with a vacuum-attached glass pipette before har-
vesting the lysis layer for analysis of the cytoplasmic proteins.
Subsequently, the residual layers and the upper half of the nuclei
22 Karl Katholnig et al.

wash layer should again be removed with the glass pipette

before harvesting the nuclei.
7. The protein concentration of the banded nuclei collected in
150 μL is usually 0.25–0.5 mg/mL depending on the cell
type. In order to measure the protein concentration or obtain
a higher concentration in the sample the banded nuclei can be
collected, diluted with 600 μL of PBS, pelleted by centrifuga-
tion at 1,000 × g, followed by protein extraction in a reduced
volume of the desired extraction buffer.

Acknowledgements

We would like to thank Werner Poglitsch for technical support in

initial experiments.

References

1. Graham JM, Rickwood D (1997) Subcellular 11. Graham JM (2001) Isolation of nuclei and
fractionation. A practical approach. Oxford nuclear membranes from animal tissues. Curr
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2. Claude A (1975) The coming of age of the cell. 12. Barta CA, Sachs-Barrable K, Feng F et al
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3. Barthelery M, Salli U, Vrana KE (2007) P-glycoprotein: modulation of the activity and
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16:905–919 13. Valenzuela SM, Martin DK, Por SB et al
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Unravelling the nuclear matrix proteome. chloride ion channel of cell nuclei. J Biol Chem
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6. Cox B, Emili A (2006) Tissue subcellular frac- 15. Hoffmann P, Chalkley R (1978) Procedures
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Protoc 1:1872–1878 Methods Cell Biol 17:1–12
7. Birnie GD (1978) Isolation of nuclei from ani- 16. Rickwood D, Messent A, Patel D (1997)
mal cells in culture. Methods Cell Biol Subcellular fractionation: a practical approach.
17:13–26 IRL Press, Oxford, pp 71–105
8. Graham J, Ford T, Rickwood D (1994) The 17. Graham JM (2002) Rapid purification of nuclei
preparation of subcellular organelles from from animal and plant tissues and cultured
mouse liver in self-generated gradients of cells. ScientificWorld J 2:1551–1554
iodixanol. Anal Biochem 220:367–373 18. Dunphy WG, Rothman JE (1985) Compart-
9. Blobel G, Potter VR (1966) Nuclei from rat mental organization of the Golgi stack. Cell
liver: isolation method that combines purity 42:13–21
with high yield. Science 154:1662–1665 19. Goldberg DE, Kornfeld S (1983) Evidence for
10. Rio DC, Ares M Jr., Hannon GJ et al (2010) extensive subcellular organization of asparagin-
Preparation of cytoplasmic and nuclear rna linked oligosaccharide processing and lyso-
from tissue culture cells. Cold Spring Harb somal enzyme phosphorylation. J Biol Chem
Protoc pdb.prot5441 258:3159–3165
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20. Dyer RB, Herzog NK (1995) Isolation of a multifunctional lysis gradient. J Immunol
intact nuclear extract preparation from a fragile Meth 373:167–173
B-lymphocyte cell line. Biotechniques 19: 23. Cimini AM, Moreno S, Giorgi M, Serafini B,
192–195 Ceru MP (1993) Purification of peroxisomal
21. Ford T, Graham J, Rickwood D (1994) fraction from rat brain. Neurochem Int 23(3):
Iodixanol: a nonionic iso-osmotic centrifuga- 249–260
tion medium for the formation of self-generated 24. Graham JM (2001) Purification of a crude
gradients. Anal Biochem 220:360–366 mitochondrial fraction by density-gradient
22. Poglitsch M, Katholnig K, Saemann MD et al centrifugation. Curr Protoc Cell Biol Chapter
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immune cells by a single centrifugation through cb0304s04
 Chapter 3

Isolation of Nuclei in Media Containing an Inert Polymer

to Mimic the Crowded Cytoplasm
Ronald Hancock and Yasmina Hadj-Sahraoui

Abstract
Within cells, the nucleus is surrounded by the cytoplasm which contains diffusible macromolecules at a
high concentration (>100 mg/ml). When cells are broken to isolate nuclei by current methods these
macromolecules are dispersed, and to reproduce the environment of nuclei in vivo more closely we have
developed a method to isolate them in a medium where cytoplasmic macromolecules are replaced by an
inert, volume-occupying polymer and which is essentially cation-free. Nuclei isolated by this method
resemble closely those prepared by conventional procedures as seen by optical and electron microscopy,
and their internal compartments (nucleoli, PML and Cajal bodies, transcription centers, and splicing
speckles) and transcriptional activity are conserved. This procedure is efficient for mammalian cells that
normally grow in suspension and do not have an extensive cytoskeleton, and requires ~30 min.

Key words Isolation of nuclei, Nuclear compartments, Macromolecular crowding, Osmotic pressure,
Ficoll, Dextran, Polyethylene glycol (PEG)

1 Introduction

Cell nuclei are commonly isolated in media containing cations at

mM concentrations [1, 2] which screen the negative charges on
chromatin and maintain its compaction [3, 4] and conserve nuclear
ultrastructure and functions. Without these cations, isolated nuclei
swell and may lyse due to the osmotic pressure caused by their high
internal concentration of macromolecules [1]. However, standard
isolation media may not reproduce the ionic environment in the
cytoplasm which surrounds the nucleus in vivo, because in the cell
K+ and Na+ ions are predominantly bound to macromolecules
[5–8] and Mg2+ ions to ATP, macromolecules, mitochondria, and
the sarcoplasmic reticulum [9, 10], and biophysical considerations
suggest that “there is no cytoplasmic bulk concentration of ions
and metabolites (as is often assumed in biophysical models and in
the design and interpretation of in vitro experiments)” [11].

Ronald Hancock (ed.), The Nucleus, Methods in Molecular Biology, vol. 1228,
DOI 10.1007/978-1-4939-1680-1_3, © Springer Science+Business Media New York 2015

25
26 Ronald Hancock and Yasmina Hadj-Sahraoui

Significant quantities of extra Mg2+ and Ca2+ ions become bound

to nuclei during their isolation in conventional media [12].
Since the cytoplasm contains diffusible macromolecules at a
concentration in the range of 130 mg/ml [13–16] the nucleus,
like other cytoplasmic organelles and macromolecular assemblies,
is predicted to experience strong macromolecular crowding effects
[17–19]. To mimic these crowding effects media containing proteins
or inert polymers have been used to isolate mitochondria [20–22],
peroxisomes [23], chloroplasts [24], Golgi apparatuses [25], and
nucleoids of bacteria [26] but similar media have been employed
only rarely to isolate nuclei [27, 28].
With these considerations in mind and recalling Arthur
Kornberg’s seventh commandment “Correct for extract dilution
with molecular crowding” [29], we have developed a method to
isolate nuclei in media in which an inert, volume-occupying poly-
mer replaces the cytoplasmic macromolecules dispersed upon cell
breakage and whose only ionic component is 100 μM K-Hepes
buffer, pH 7.4. This environment is predicted to reproduce that of
nuclei in vivo more closely than conventional isolation media. The
polymers used are Ficoll or dextran of Mr 70 kDa, molecules of a
size excluded from nuclei [30, 31] but which are inconvenient to
handle and pipette due to the viscosity of their solutions, or poly-
ethylene glycol (PEG) of Mr 8 kDa which penetrates through the
nuclear envelope and exerts crowding effects on macromolecules
and structures within the nucleus; PEG forms solutions of low
viscosity which are convenient to use and provides nuclei with
similar properties [31]. The cytoplasmic membrane is permeabilized
by the non-ionic detergent digitonin, which releases cytoplasmic
material while conserving the semipermeable properties of the
nuclear membrane [32]. These nuclei are free of cytoplasmic material
and closely resemble those prepared by conventional methods, as
seen by optical and electron microscopy (Fig. 1), and their internal
compartmentalization (Fig. 2) and transcriptional activity [31] are
conserved, consistent with the idea that crowding by cytoplasmic
macromolecules is an important but overlooked factor which
determines the structure and functions of nuclei in vivo. This pro-
tocol is efficient for cell types that normally grow in suspension and
do not have an extensive cytoskeleton, and requires ~30 min.

2 Materials

2.1 Cells This method has been used successfully for cells of the lines Raji
(human lymphoblastoid, ATCC CCL-86), K562 (human erythro-
leukemia, ATCC CCL-243), and P815 (mouse mastocytoma,
ATCC TIB-64) and for human lymphocytes (see Note 1). Cells are
harvested during exponential growth.
 Nuclear Isolation in Crowded Media 27

Fig. 1 Nuclei of K562 cells released in polymer solution containing 100 μM K-Hepes and digitonin. These nuclei
are free of cytoplasmic material and closely resemble those prepared by conventional methods. (a–d) phase
contrast images of (a) intact cells; (b–d) nuclei centrifuged onto slides after (b) homogenizing cells in 12 kDa
PEG; (c) vortexing cells in 70 kDa Ficoll; (d) vortexing cells in 70 kDa dextran. (e–g) electron microscope
sections of (e) intact cells; (f) nuclei isolated in PEG; (g) nuclei isolated in Ficoll. Sections were stained with
uranyl acetate and lead citrate. Scale bars a–d, f, g = 10 μm, e = 1 μm. Modified from [31]

2.2 Reagents 1. 100 mM K-Hepes buffer (pH 7.4): to prepare 100 ml dissolve
2.38 g of Hepes in bidistilled H2O. Adjust the solution to
pH 7.4 by careful dropwise addition of 100 mM KOH while
mixing on the pH meter, and filter through a 0.45 μm mem-
brane filter. Store aliquots at –20 °C.
2. Polymer solutions: to prepare 50 ml of 12 % (w/v) 8 kDa PEG,
50 % (w/v) 70 kDa Ficoll, or 35 % (w/v) 70 kDa dextran,
respectively, weigh 6 g of PEG (average Mr 8 kDa, Fluka
molecular biology grade), 25 g of Ficoll (average Mr 70 kDa,
Fluka), or 17.5 g of dextran (average Mr 69.9 kDa, Sigma-
Aldrich) into a 50 ml conical polypropylene tube.
Dissolve the polymer in bidistilled H2O by incubating the
solution in a thermostat at 60–70 °C with intermittent vortex-
ing; PEG requires 1–2 h to dissolve while Ficoll and dextran
may need several hours and vigorous vortexing. Cool the solu-
tion to room temperature and deionize by adding ~1 g of
mixed-bed ion-exchange resin (AG 501-X8D, Bio-Rad) and
mix occasionally over 6–8 h. Allow the resin beads to settle
and pass the solution through an 0.45 μm syringe filter; dex-
tran and Ficoll solutions filter slowly. Add 1/1,000 volume of
100 mM K-Hepes buffer (pH 7.4), verify the pH, and if nec-
essary adjust to pH 7.4 by adding μl volumes of 100 mM
28 Ronald Hancock and Yasmina Hadj-Sahraoui

Fig. 2 Compartments are conserved in nuclei isolated in polymer solution. (a, b) Nuclei of K562 cells
centrifuged onto slides and labeled with primary antibodies to the protein indicated followed by an appropriate
secondary antibody conjugated with Alexa 488, 568, or 594, in order to visualize nucleoli, PML and Cajal bodies,
transcription centers (RNA polymerase II), or splicing (SC35) speckles [33]. (a) Nuclei isolated in 12 kDa PEG
or (b) in 70 kDa Ficoll. (c) Localization of coilin-GFP or PML isoform IV-GFP in nuclei of HeLa or U2OS cells,
respectively, which express these proteins. Cells grown on slides were washed and permeabilized and
extracted in situ with 70 kDa Ficoll solution containing digitonin (see Note 1). Images are maximum intensity
projections from serial 0.5 μm confocal sections; DNA was stained with DAPI. The numbers of nucleoli, PML
bodies, and Cajal bodies are essentially identical to those in nuclei isolated in a conventional ionic medium
[31]. Scale bars = 5 μm. Modified from [31]

KOH while mixing on the pH meter. Store at 4 °C and verify

the pH before each experiment using a plastic pH indicator
strip (see Note 2).
3. Digitonin solution: dissolve digitonin in bidistilled H2O at
10 mg/ml by incubation for 15 min at 98 °C; we use Boehringer
high purity digitonin which is no longer available, but
high-purity digitonin from other suppliers provides satisfactory
 Nuclear Isolation in Crowded Media 29

results. Cool the solution and store in aliquots at 4 °C; if a

precipitate forms, redissolve by heating the solution in the same
conditions.
4. Polymer solution supplemented with digitonin (100 μg/ml):
prepare freshly for each experiment.
5. Two ml volume glass homogenizer with teflon piston (Potter-
Elvehjem Type Tissue Grinder; Wheaton, Millville, NJ, USA).
6. Microscope slides coated with polylysine: these can be purchased,
or alternatively dip slides in a 50 μg/ml solution of poly-L
lysine for ~1 h, wash with deionized H2O, drain well, and allow
to dry in air.

3 Methods

Ficoll and dextran solutions at concentrations which maintain the

volume of isolated nuclei are viscous and inconvenient to pipette.
Solutions containing 12 % 8 kDa PEG are much less viscous and
more convenient to use, and produce similar results. The buffering
capacity of these solutions is low and their pH must be checked
before each experiment. As described here, the method provides
sufficient nuclei for imaging studies but it can be scaled up by
increasing the volumes of solutions in proportion to the number of
cells. All steps are carried out at room temperature, and nuclei are
handled using micropipettes with ~5 mm cut from the tip.
1. To isolate nuclei in Ficoll or dextran, it is not practical to
wash intact cells by centrifugation because of the high density
and viscosity of these solutions. Instead, centrifuge ~107
cells a 15 ml conical polypropylene tube, remove all growth
medium carefully with a micropipette and then with an
absorbent paper wick, and resuspend the cells in 1 ml of
polymer solution containing digitonin. After 10 min mix on
a vortexer at maximum speed until >95 % of the cells have
released their nucleus (usually 2–3 min); examine by phase-
contrast microscopy after placing 5 μl samples on slides and
covering with a cover glass. Add two volumes of the same
polymer solution without digitonin to the suspension of
nuclei and mix by inverting the tube.
2. To isolate nuclei in PEG solution, centrifuge ~107 cells in a
15 ml conical polypropylene tube, remove all the medium
using a pipette attached to a vacuum line, and resuspend the
cells in 1 ml of PEG solution by hand mixing. Centrifuge the
cells (600 × g, 10 min), resuspend by gentle hand mixing in
1 ml of PEG solution containing digitonin, and transfer the
suspension to a 2 ml glass homogenizer with a teflon piston
After 10 min, homogenize using slow hand strokes until >95 %
30 Ronald Hancock and Yasmina Hadj-Sahraoui

of the cells have released their nucleus, avoiding foaming

(usually ~50 strokes); examine by phase-contrast microscopy
(see Subheading 3, step 1). Add two volumes of the same PEG
solution without digitonin and mix.
3. For optical (Fig. 1) or fluorescence imaging (Fig. 2) of nuclei,
mount polylysine-coated slides in a cytological centrifuge,
transfer aliquots of the suspension containing ~106 nuclei into
the slide chambers, and centrifuge the nuclei onto the slides at
4,000 × g for 40 min in Ficoll or dextran or 1,000 × g for 10 min
in PEG. For fixation, remove the solution by tapping the slides
edge-wise on absorbent paper, overlay the nuclei with 0.5 ml
of the same polymer solution used for the previous step supple-
mented with the desired fixative (we fix with 2 % (w/v)
paraformaldehyde for 10 min). Incubate the slides in a humid-
ified container (see Note 3).
4. If the nuclei must be washed for subsequent experiments,
centrifuge them at 5,000 × g for 15 min in Ficoll or dextran or
2,000 × g for 5 min in PEG, resuspend them in the same polymer
solution without digitonin, and centrifuge them in the same
conditions (see Notes 4–6).

4 Notes

1. Fibroblastoid cells (e.g., HeLa, CHO, or U2OS cells) do not

yield clean nuclei by this procedure because fibrous extracel-
lular and cytoskeletal materials sediment with the nuclei and
cannot be removed. Instead, these cells can be grown on
slides or cover glasses and extracted in situ with the same
solutions, leaving the nuclei attached to the surface. Remove
all growth medium from slides or cover glasses carefully
using a micropipette and absorbent paper wicks, overlay the
cells with 500 μl of polymer solution for 5 min to wash them,
and replace this solution with 500 μl of the same solution
containing digitonin for 30 min to permeabilize and extract
them (Fig. 2c).
2. PEG solutions may develop a slight yellow colour and their
pH may decrease after storage for several weeks, due to uniden-
tified processes, but this does not affect their efficacy in this
method detectably.
3. A suitable humid chamber is a sealable plastic box containing
a support to carry slides horizontally and moistened paper
towels at the bottom.
4. If it is desired to examine the nuclei by electron microscopy,
they can be centrifuged (5,000 × g for 15 min in Ficoll or
dextran or 2,000 × g for 5 min in PEG) and resuspended in the
 Nuclear Isolation in Crowded Media 31

same polymer solution containing fixative; we use 2 % (w/v)

paraformaldehyde with 0.1 % (v/v) glutaraldehyde (both EM
grade, Ted Pella, Redding, CA, USA) for 1 h on ice. The fixed
nuclei can be centrifuged, embedded in 1.5 % (w/v) low
melting-point agarose, dehydrated, embedded in an appropriate
resin, sectioned, and stained with uranyl acetate and lead citrate
(Fig. 1e–g) [31].
5. For further studies of isolated nuclei, for example of
transcription or DNA replication, their osmotic conditions
and morphology must be conserved by working in the same
polymer solution used to isolate them.
6. Metaphase chromosomes can be isolated from mitotic cells in
the same conditions [34].

Acknowledgements

We thank Jason Swedlow (Wellcome Trust Biocentre, University

of Dundee, Scotland) for HeLa cells expressing GFP-coilin and
David Bazett-Jones (Hospital for Sick Children, Toronto, Canada)
for U2OS cells expressing GFP-PML isoform IV (originally from
J. Taylor, Medical College of Wisconsin).

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