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Neuromethods 144
Yuji Odagaki
Dasiel O. Borroto-Escuela Editors
Co-Immuno-
precipitation
Methods
for Brain Tissue
Neuromethods
Series Editor
Wolfgang Walz
University of Saskatchewan
Saskatoon, SK, Canada
For further volumes:

http://www.springer.com/series/7657
Co-Immunoprecipitation Methods
for Brain Tissue
Edited by
Yuji Odagaki
Department of Psychiatry, Saitama Medical University, Saitama, Japan
Dasiel O. Borroto-Escuela
Department of Neuroscience, Karolinska Institutet, Stockholm, Sweden
Editors
Yuji Odagaki Dasiel O. Borroto-Escuela
Department of Psychiatry Department of Neuroscience
Saitama Medical University Karolinska Institutet
Saitama, Japan Stockholm, Sweden
ISSN 0893-2336 ISSN 1940-6045 (electronic)

Neuromethods
ISBN 978-1-4939-8984-3 ISBN 978-1-4939-8985-0 (eBook)
https://doi.org/10.1007/978-1-4939-8985-0
Library of Congress Control Number: 2018962873
© Springer Science+Business Media, LLC, part of Springer Nature 2019

This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of the material is
concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction
on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation,
computer software, or by similar or dissimilar methodology now known or hereafter developed.
The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not
imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and
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The publisher, the authors, and the editors are safe to assume that the advice and information in this book are believed
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This Humana Press imprint is published by the registered company Springer Science+Business Media, LLC, part of
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The registered company address is: 233 Spring Street, New York, NY 10013, U.S.A.
Preface to the Series
Experimental life sciences have two basic foundations: concepts and tools. The Neuromethods
series focuses on the tools and techniques unique to the investigation of the nervous system
and excitable cells. It will not, however, shortchange the concept side of things as care has
been taken to integrate these tools within the context of the concepts and questions under
investigation. In this way, the series is unique in that it not only collects protocols but also
includes theoretical background information and critiques which led to the methods and
their development. Thus, it gives the reader a better understanding of the origin of the
techniques and their potential future development. The Neuromethods publishing program
strikes a balance between recent and exciting developments like those concerning new
animal models of disease, imaging, in vivo methods, and more established techniques,
including, for example, immunocytochemistry and electrophysiological technologies. New
trainees in neurosciences still need a sound footing in these older methods in order to apply
a critical approach to their results.
Under the guidance of its founders, Alan Boulton and Glen Baker, the Neuromethods
series has been a success since its first volume published through Humana Press in 1985.
The series continues to flourish through many changes over the years. It is now published
under the umbrella of Springer Protocols. While methods involving brain research have
changed a lot since the series started, the publishing environment and technology have
changed even more radically. Neuromethods have the distinct layout and style of the
Springer Protocols program, designed specifically for readability and ease of reference in a
laboratory setting.
The careful application of methods is potentially the most important step in the process
of scientific inquiry. In the past, new methodologies led the way in developing new
disciplines in the biological and medical sciences. For example, physiology emerged out of
anatomy in the nineteenth century by harnessing new methods based on the newly
discovered phenomenon of electricity. Nowadays, the relationships between disciplines and
methods are more complex. Methods are now widely shared between disciplines and
research areas. New developments in electronic publishing make it possible for scientists
that encounter new methods to quickly find sources of information electronically. The
design of individual volumes and chapters in this series takes this new access technology
into account. Springer Protocols makes it possible to download single protocols separately.
In addition, Springer makes its print-on-demand technology available globally. A print copy
can therefore be acquired quickly and for a competitive price anywhere in the world.
Saskatoon, SK, Canada Wolfgang Walz
v
Preface
The human genome consists of 20,000–30,000 genes coding for 100,000–500,000

different proteins, of which more than 10,000 proteins can be produced by the cell at any
given time. Interestingly, it has been recognized that most proteins do not function on
their own but as part of large signaling complexes that are arranged in every living cell in
response to specific environmental cues. For instance, in the brain, nearly all functions
require the existence of multiprotein complexes; thus, the identification and c haracterization
of protein- protein interactions (PPIs) constitutes an essential step to understand its
functioning. Consequently, PPI discovery and characterization in different types of brain
cells may provide useful information to understand the complexity of brain.
Several methods can be applied for discovery, validation, and characterization of PPI
processes. Importantly, before implementing any PPI method, two fundamental notions
have to be kept in mind: (1) sensitivity and (2) specificity. In such way, if the objective is to
detect as many as possible life-occurring PPIs, a high-sensitivity screen methodology is
needed. However, if the goal is to achieve a high PPI validation rate, a high specificity
method should be chosen, thus ensuring that most of the interactions detected by the
screen method will occur in life. Accordingly, since all PPI methods have its own strengths
and weaknesses, a balance between sensitivity and specificity should be accomplished when
choosing a concrete PPI method.
PPI methods can be classified into two main categories, namely, biochemical approaches
and biophysical (including theoretical) technologies. Within the biochemical methods, the
co-immunoprecipitation (Co-IP) is considered the gold standard PPI assay, especially when
endogenous PPIs (i.e., not overexpressed and not tagged proteins) are assessed. Co-IP is a
simple but effective PPI method that relies on target-specific antibodies.
Co-IP is conducted in essentially the same manner as an immunoprecipitation, except
that the target antigen precipitated by the antibody is used to coprecipitate its binding
partner(s) or associated protein complex from the lysate. When associated proteins are
coprecipitated, it is usually assumed that these associated proteins are related to the f unction
of the target antigen at the cellular level. As such, Co-IP is frequently used as the initial
experiments in a tedious and protracted experimental process with ultimate purpose of
identifying functional interactions of a protein of interest.
Co-IP possesses several advantages. The results are highly reproducible, and the
assay is relatively inexpensive. However, these methods also have some disadvantages
or limitations. Immunoprecipitation as it is normally performed does not provide
quantitative data regarding the affinity or stoichiometry of an interaction. Co-IP for
complex mixture instead of purified proteins does not differentiate between direct and
indirect protein-protein interactions. Also, the variability among proteins and the
myriad factors that can affect protein structure and interaction leads to potential
problem as to the experimental conditions for immunoprecipitation. The protocols for
Co-IP have to be optimized individually according to the sample, target protein(s),
antibody used, and so on.
vii
viii Preface
The contributors to this book cover all these aspect of Co-IP methods and their relevant
use to study PPIs in health and diseases of the CNS. They present the state of the art of
Co-IP assay, also excellent and updated “Notes” about the optimization and t roubleshooting
of this technique. These protocols have been carefully detailed by specialists in the field, and
their work remains current and of great interest to researchers of many years to come.
Release of this volume on Co-Immunoprecipitation Method for Brain Tissue is therefore
timely.
Saitama, Japan Yuji Odagaki

Stockholm, Sweden Dasiel O. Borroto-Escuela
Contents
Preface to the Series . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v

Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vii
Contributors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xi
1 Co-immunoprecipitation Methods for Detection of G Protein-Coupled

Receptors in Brain Tissue�� 1
Kazunori Namba and Hiroki Kaneko
2 Co-immunoprecipitation Methods to Identify Associated Proteins
with Estrogen Receptor α at Postsynaptic Density in Brain Tissue �� 9
Gen Murakami and Suguru Kawato
3 The Use of Co-immunoprecipitation to Study Conformation-Specific
Protein Interactions of Oligomeric α-Synuclein Aggregates �� 23
Cristine Betzer, Rikke Hahn Kofoed, and Poul Henning Jensen
4 Using Co-immunoprecipitation and Shotgun Mass Spectrometry
for Protein-Protein Interaction Identification in Cultured
Human Oligodendrocytes�� 37
Bradley Smith, Daniel Martins-de-Souza, and Mariana Fioramonte
5 Co-immunoprecipitation Analysis of GPCR Complexes
in the Central Nervous System�� 49
Yuji Kamikubo and Takashi Sakurai
6 The Histoblot Technique: A Reliable Approach to Analyze Expression
Profile of Proteins and to Predict Their Molecular Association�� 65
Carolina Aguado and Rafael Luján
7 Co-immunoprecipitation Assay to Investigate the Interaction Strength
Between Synaptic Proteins Using COS-7 Cells�� 89
Sosuke Yagishita
8 Guanosine-5′-O-(3-[35S]thio)triphosphate ([35S]GTPγS) Binding/
Immunoprecipitation Assay Using Magnetic Beads Coated
with Anti-Gα Antibody in Mammalian Brain Membranes�� 97
Yuji Odagaki
9 Co-immunoprecipitation as a Useful Tool for Detection
of G Protein-Coupled Receptor Oligomers �� 109
Kirill Shumilov, Alejandra Valderrama-Carvajal, María García-Bonilla,
and Alicia Rivera
ix
x Contents
10 Isolation and Detection of G Protein-Coupled Receptor (GPCR)

Heteroreceptor Complexes in Rat Brain Synaptosomal Preparation
Using a Combined Brain Subcellular Fractionation/Co-immunoprecipitation
(Co-IP) Procedures�� 123
Dasiel O. Borroto-Escuela, Manuel Narvaez, Martina Zannoni,
Chiara Contri, Minerva Crespo-Ramírez, Michael di Palma,
Patrizia Ambrogini, Daily Y. Borroto-Escuela, Ismel Brito,
Mariana Pita-Rodríguez, Ismael Valladolid-Acebes, Miguel Pérez de la
Mora, and Kjell Fuxe
11 Co-immunoprecipitation of Membrane-Bound Receptors
from Subsynaptic Compartments�� 137
Wilber Romero-Fernandez, Maria Garcia-Barcelo,
and Yunis Perez-Betancourt
12 Coimmunoprecipitation (co-IP) Analysis for Protein-Protein
Interactions in the Neurons of the Cerebral Ganglia
of the Land Snails of the Genus Polymita During Aestivation�� 147
Daily Y. Borroto-Escuela, Idania Hernández-Ramos, Kjell Fuxe,
and Dasiel O. Borroto-Escuela
13 Co-immunoprecipitation (Co-IP) of G Protein-Coupled
Receptor (GPCR)-Receptor Tyrosine Kinase (RTK)
Complexes from the Dorsal Hippocampus of the Rat Brain�� 157
Michael Di Palma, Manuel Narvaez, Mariana Pita-Rodríguez,
Chiara Contri, Martina Zannoni, Riccardo Cuppini, Kjell Fuxe,
Patrizia Ambrogini, and Dasiel O. Borroto-Escuela
Index�� 165
Contributors
Carolina Aguado • Facultad de Medicina, Departamento de Ciencias Médicas, Instituto

de Investigación en Discapacidades Neurológicas (IDINE), Universidad Castilla-La
Mancha, Albacete, Spain
Patrizia Ambrogini • Section of Physiology, Department of Biomolecular Science,
University of Urbino, Urbino, Italy
Cristine Betzer • Danish Research Institute of Translational Neuroscience—
DANDRITE, Aarhus University, Aarhus, Denmark; Department of Biomedicine,
Aarhus University, Aarhus, Denmark
Dasiel O. Borroto-Escuela • Department of Neuroscience, Karolinska Institutet,
Stockholm, Sweden; Observatorio Cubano de Neurociencias, Yaguajay, Cuba
Daily Y. Borroto-Escuela • Observatorio Cubano de Neurociencias, Yaguajay, Cuba;
Environmental Services Center, Caguanes National Park, Ministry of Science, Technology
and Environment (CITMA), Yaguajay, Cuba
Ismel Brito • Department of Neuroscience, Karolinska Institutet, Stockholm, Sweden;
Observatorio Cubano de Neurociencias, Yaguajay, Cuba
Chiara Contri • Department of Neuroscience, Karolinska Institutet, Stockholm, Sweden;
Department of Life Science and Biotechnology, University of Ferrara, Ferrara, Italy
Minerva Crespo-Ramírez • Instituto de Fisiología Celular, Universidad Nacional
Autónoma de México, Mexico City, Mexico
Riccardo Cuppini • Section of Physiology, Department of Biomolecular Science,
Miguel Pérez de la Mora • Instituto de Fisiología Celular, Universidad Nacional
Autónoma de México, Mexico City, Mexico
Michael di Palma • Section of Physiology, Department of Biomolecular Science,
Mariana Fioramonte • Laboratory of Neuroproteomics, Department of Biochemistry and
Tissue Biology, Institute of Biology, University of Campinas (UNICAMP), Campinas,
Brazil
Kjell Fuxe • Department of Neuroscience, Karolinska Institutet, Stockholm, Sweden
Maria Garcia-Barcelo • Faculty of Health Sciences, Technical University of Ambato,
Ambato, Ecuador
María García-Bonilla • Facultad de Ciencias, Universidad de Málaga, Instituto de
Investigación Biomédica, Málaga, Spain
Idania Hernández-Ramos • Environmental Services Center, Caguanes National Park,
Ministry of Science, Technology and Environment (CITMA), Yaguajay, Cuba
Poul Henning Jensen • Danish Research Institute of Translational Neuroscience—
Yuji Kamikubo • Department of Pharmacology, Juntendo University School of Medicine,
Tokyo, Japan
xi
xii Contributors
Hiroki Kaneko • Laboratory of Nuclear Transport Dynamics, National Institutes of

Biomedical Innovation, Health and Nutrition (NIBIOHN), Osaka, Japan; The
Institute of Natural Sciences, College of Humanities and Sciences, Nihon University,
Tokyo, Japan
Suguru Kawato • Department of Biophysics and Life Sciences, Graduate School of Arts
and Sciences, University of Tokyo, Tokyo, Japan; Department of Urology, Graduate School
of Medicine, Juntendo University, Tokyo, Japan; Faculty of Pharma-Science, Department
of Cognitive Neuroscience, Teikyo University, Tokyo, Japan
Rikke Hahn Kofoed • Danish Research Institute of Translational Neuroscience—
Rafael Luján • Facultad de Medicina, Departamento de Ciencias Médicas, Instituto de
Investigación en Discapacidades Neurológicas (IDINE), Universidad Castilla-La
Mancha, Albacete, Spain
Daniel Martins-de-Souza • Laboratory of Neuroproteomics, Department of Biochemistry
and Tissue Biology, Institute of Biology, University of Campinas (UNICAMP),
Campinas, Brazil; Instituto Nacional de Biomarcadores em Neuropsiquiatria
(INBION), Conselho Nacional de Desenvolvimento Cientifico e Tecnologico, Sao Paulo,
Brazil
Gen Murakami • Faculty of Medicine, Department of Liberal Arts, Saitama Medical
University, Saitama, Japan
Kazunori Namba • Division of Hearing and Balance Research, National Institute of
Sensory Organs, National Hospital Organization Tokyo Medical Center, Tokyo, Japan
Manuel Narvaez • Facultad de Medicina, Instituto de Investigación Biomédica de
Málaga, Universidad de Málaga, Málaga, Spain
Yuji Odagaki • Faculty of Medicine, Department of Psychiatry, Saitama Medical
University, Saitama, Japan
Yunis Perez-Betancourt • Faculty of Food Science and Engineering, Technical
University of Ambato, Ambato, Ecuador
Mariana Pita-Rodríguez • Facultad de Medicina, Instituto de Investigación Biomédica
de Málaga, Universidad de Málaga, Málaga, Spain; Neurogenetics Department,
Institute of Neurology and Neurosurgery, Havana, Cuba
Alicia Rivera • Facultad de Ciencias, Universidad de Málaga, Instituto de Investigación
Biomédica, Málaga, Spain
Wilber Romero-Fernandez • Department of Cell and Molecular Biology, Uppsala
University, Uppsala, Sweden; Faculty of Health Sciences, Technical University of Ambato,
Ambato, Ecuador
Takashi Sakurai • Department of Pharmacology, Juntendo University School of Medicine,
Tokyo, Japan
Kirill Shumilov • Facultad de Ciencias, Universidad de Málaga, Instituto de
Investigación Biomédica, Málaga, Spain
Bradley Smith • Laboratory of Neuroproteomics, Department of Biochemistry and Tissue
Biology, Institute of Biology, University of Campinas (UNICAMP), Campinas, Brazil
Alejandra Valderrama-Carvajal • Facultad de Ciencias, Universidad de Málaga,
Instituto de Investigación Biomédica, Málaga, Spain
Contributors xiii
Ismael Valladolid-Acebes • The Rolf Luft Research Center for Diabetes and
Endocrinology, Karolinska Institutet, Karolinska University Hospital L1, Stockholm,
Sweden
Sosuke Yagishita • Department of Peripheral Nervous System Research, National
Institute of Neuroscience, National Center of Neurology and Psychiatry, Tokyo, Japan
Martina Zannoni • Department of Neuroscience, Karolinska Institutet, Stockholm,
Sweden; Department of Life Science and Biotechnology, University of Ferrara, Ferrara,
Italy
Chapter 1
Co-immunoprecipitation Methods for Detection

of G Protein-Coupled Receptors in Brain Tissue
Kazunori Namba and Hiroki Kaneko
Abstract
Some G protein-coupled receptors (GPCRs) modify their own signaling by interacting with other
GPCR types. In brain tissue, co-immunoprecipitation is one of the most effective method by which to
detect GPCR oligomers. Evidence of the direct association of molecules in brain tissue is desirable as
in vivo conditions more closely mimic the natural environment than do in vitro studies. GPCR
hetero-oligomerization may contribute to the unexpected effects of new GPCR-targeted drugs, making
detection of these assemblies in vivo important for drug discovery. However, GPCR expression is
generally lower in brain tissue compared with that in in vitro experiments, as promoters are used to
artificially drive GPCR expression; consequently, less heteromeric interaction is detectable in brain
tissue. Therefore, there are few reports of GPCR heteromeric oligomerization containing

co-immunoprecipitation data from brain tissue.
In this chapter, we introduce an effective method for co-immunoprecipitation of GPCRs using rodent
brain tissue. We modified a general protocol to obtain direct evidence of GPCR interaction in the brain.
Key words Co-immunoprecipitation, Immunoprecipitation, Pull down, Brain tissue, GPCR
1 Introduction
It is known that a significant number of G protein-coupled

r eceptors (GPCRs) can form dimers or higher-order oligomer
complexes, which can have various functions [1]. Many GPCRs
exist as heteromeric assemblies (referred to as
hetero-oligomerization), which modifies their ligand binding
and second messenger functions [2], and in turn creates unique
r eceptor trafficking systems for pharmacological profiles [3, 4].
Because most GPCRs are expressed in neural cells, brain tissue
has been targeted to help elucidate GPCR cross talk, particu-
larly in the olfactory bulb, where many types of GPCRs are
concentrated [5].
In our laboratory, we attempted to elucidate cross talk between
several types of purinergic receptors (P1 and P2) [6]. Purines such
Yuji Odagaki and Dasiel O. Borroto-Escuela (eds.), Co-Immunoprecipitation Methods for Brain Tissue, Neuromethods, vol. 144,
https://doi.org/10.1007/978-1-4939-8985-0_1, © Springer Science+Business Media, LLC, part of Springer Nature 2019
1
2 Kazunori Namba and Hiroki Kaneko
as adenosine triphosphate (ATP), via their specific P1 and P2

receptors, mediate a variety of physiological processes, including
pathophysiology, neurotransmission, neuromodulation, pain,
cardiac function, immune responses, and many aspects of

development [7–10]. P1 receptors are further classified into A1,
A2A, A2B, and A3 sub-types, all of which are GPCRs. P2 receptors
can be further classified into ligand-gated ion channel-type P2X
[1–7] receptors and G protein-coupled P2Y [1, 2, 4, 6, 11–14]
receptors. The adenosine A1 receptor (A1R) is known to regulate
Ca2+/K+ channels, adenylate cyclase, and phospholipase C by
coupling to Gi/o proteins [10].
In our previous study, we found that A1R associated with
P2Y1R in co-transfected HEK293T cells was substantially modified
when a P2Y1R agonist stimulated A1R signaling via Gi/o [11].
Furthermore, in cultured cells co-transfected with A1R and P2Y2R,
hetero-oligomers showed synergistic increases in Ca2+ signaling,
whereby simultaneous activation of the two receptors attenuated
A1R signaling via Gi/o but synergistically enhanced P2Y2R signaling
via Gq/11 [12]. In these studies, we demonstrated the hetero-
oligomerization of these GPCRs using co-immunoprecipitation
methods in cultured cells.
For the next stage, we demonstrated evidence of an association
between A1R and P2Y1R in rat brain by immunohistochemical
study and co-immunoprecipitation using brain tissue homogenate
[13]. Other experiments using co-immunoprecipitation methods
targeted detection of A1R and purinergic P2Y2R hetero-
oligomerization [14, 15]. These experiments allowed us to improve
our co-immunoprecipitation technique in brain tissue.
Co-immunoprecipitation data from brain tissue could help to
explain hetero-oligomerization ex vivo and at endogenous e xpression
levels in addition to that observed in transfected cells. During drug
screening, data from several experimental environments are
important because they provide information for each stage of drug
discovery [16].
Despite the importance of direct evidence of GPCR assemblies
in vivo, co-immunoprecipitation data from brain tissue (endogenous
protomer expression level) are poor compared with data from gene
transfection studies in cultured cells (heterologous protomer expres-
sion). The following three reasons may account for the poor data.
One reason is that gene expression is lower in brain tissue compared
with that in cultured cells, where a transgene expression vector is
induced to express the cloned gene by the corresponding promoter.
Another reason is the difficulty of comparatively evaluating quantity
because the expression levels of each gene vary by brain region.
Finally, identifying target proteins is difficult when there are many
heterogeneous cells like those in brain tissue; c ultured cells are more
Co-Immunoprecipitation of GPCRs in Brain Tissue 3
homogeneous, making it easier to focus on the target genes.

Compared with transfected cell cultures, non-specific binding may
occur in brain tissue homogenate co-immunoprecipitation experi-
ments. Therefore, some brain tissue co-immunoprecipitation experi-
ments may fail. Despite these d ifficulties, we aimed to detect the
direct interaction of molecules via several modifications to a general
brain tissue co-immunoprecipitation protocol. In this chapter, we
introduce an effective co-immunoprecipitation methods for the
study of GPCR-GPCR interactions in the brain tissue, using A1R
and P2Y2R as an example.
2 Materials
2.1 Animal Eight-week-old Wistar rats were anesthetized with pentobarbital

Preparation (30 mg/kg, intravenous [i.v.]) and decapitated. The brains were
removed, and the cortex, hippocampus, caudate putamen, and
cerebellum were dissected out. Sex does not seem to affect A1 and
P2Y receptor expression patterns (data not shown); therefore we
perform all experiments using male rats because their brain weight
is slightly greater.
2.2 Immuno- The tissues were homogenized with a Polytron® homogenizer in

precipitation 50 mM Tris-acetate, pH 7.4, containing a protease inhibitor
and Western Blotting cocktail (Roche Applied Science, Manheim, Germany). The

resulting cell suspensions were centrifuged at 30,000 × g for

30 min at 4 °C. The pellets were solubilized in ice-cold lysis buffer

(50 mM Tris–HCl, pH 7.4, 1% Triton X-100, 300 mM NaCl, and
a protease inhibitor cocktail) for 60 min at 4 °C. The mixture was
centrifuged at 18,500 × g for 20 min at 4 °C, and the supernatant
was precleared with Protein G Sepharose™ 4 Fast Flow (Amersham
Bioscience, Piscataway, NJ). The lysate was incubated with a rabbit
polyclonal A1R antibody (anti-A1R; 1 μg/mL; Sigma-Aldrich, St.
Louis, MO) for 60 min at 4 °C. Protein G Sepharose™ was added
to the mixture, and the incubation continued for an additional
120 min. Protein G Sepharose™ was recovered by centrifugation
and washed 3 times with lysis buffer. Immunoprecipitates were
eluted with sodium dodecyl sulfate polyacrylamide gel electropho-
resis (SDS-PAGE) sample buffer, resolved by 12% SDS-PAGE, and
electrotransferred to nitrocellulose membranes. Receptors were
detected on the blot using anti-A1R or anti-P2Y2R, followed by a
horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG
secondary antibody (Sigma-Aldrich). The reactive bands were
visualized with enhanced chemiluminescent substrates
(SuperSignal® West Pico; Pierce, Rockford, IL).
3 Methods
3.1 Co-immuno- In our reported methods, we had examined whether A1R and
precipitation of A1R P2Y2R were associated with one another in separate brain regions
and P2Y2R from Rat using co-immunoprecipitation with anti-A1R followed by immu-
Brain noblotting with both A1R and P2Y2R antibodies [13]. A1R and
P2Y2R immunoreactivities were present in the rat brain regions
examined (Fig. 1a, b, f). Moreover, in these same regions, anti-
A1Rs were capable of co-precipitating with P2Y2R (Fig. 1d), indi-
cating that A1R and P2Y2R are associated with one another in rat
cortex, cerebellum, and hippocampus. The absence of these immu-
noreactive bands in the presence of anti-P2Y2R antigen peptides
(Fig. 1c, e) is evidence of antibody specificity. It was confirmed that
immunoprecipitation in the absence of a primary antibody resulted
in a lack of detectable receptor bands in the immunoblot. Because
no antigen peptide was available for A1R, anti-A1R specificity was
confirmed by immunocytochemistry of mock-transfected
HEK293T cells: no specific band was detected (data not shown).
Fig. 1 Co-immunoprecipitation of adenosine A1 receptor (A1R) and purinergic P2Y2 receptor (P2Y2R) from the
cerebral cortex [1], cerebellum [2], hippocampus [3], and caudate putamen [4]. Immunoblotting analyses of rat
brain extracts with anti-A1R (a), anti-P2Y2R (b), and anti-P2Y2R with a P2Y2R control peptide (c). Membrane
extracts from each region were immunoprecipitated with anti-A1R and analyzed by immunoblotting with anti-
P2Y2R (d), anti-P2Y2R with the P2Y2R control peptide (e), and anti-A1R (f). No bands, indicating the specificity
of the antibodies (c, e)
3.2 Refined Conditions The amount of A1R and P2Y2R expressed in a single brain might
for Co-immuno- be too small to detect via Western blotting in co-
precipitation immunoprecipitation experiments. To increase the Western blot-
ting signal, we optimized the conditions of the original protocol
with the four changes described as follows.
1. We used 12 rat brains per experiment to maximize the co-
immunoprecipitation signal. Approximately ten rat (or mouse)
littermates are ideal, but animals of the same age and strain
could be used if littermates are unavailable.
2. To optimize the molecular interaction between GPCRs and
protein G sepharose, we exsanguinated anesthetized rats via
cardiac perfusion with phosphate buffered saline (pH 7.2).
The co-immunoprecipitation signal intensity was greater fol-
lowing exsanguination compared with the signal intensity
observed following decapitation (i.e., blood present in the
brain tissue) (Fig. 2).
Fig. 2 Co-immunoprecipitation results with the improved methods, including exsanguination. Co-immuno
precipitation of A1R in the cerebral cortex [1], cerebellum [2], hippocampus [3], and caudate putamen [4].
Immunoblotting analyses of rat brain extracts with anti-A1R (a), anti-P2Y2R (b), and anti-P2Y2R with a P2Y2R
control peptide (c). Membrane extracts were immunoprecipitated with anti-A1R and analyzed by immunoblot-
ting with anti-P2Y2R (d), anti-P2Y2R with a P2Y2R control peptide (e), and anti-A1R (f). Immunoblotting analyses
of rat brain extracts after decapitation (i.e., blood present in the samples) with anti-A1R (a′). Membrane extracts
from blood-containing samples were immunoprecipitated with anti-A1R and analyzed by immunoblotting with
anti-P2Y2R (d′)
3. To avoiding catalytic dissociation by protease during tissue

homogenization, we added protease inhibiter cocktail at
approximately 3 times the concentration of that used under
normal conditions.
4. We used an improved lysis buffer (50 mM Tris–HCl, pH 7.4,
5% Triton X-100, 300 mM NaCl, and protease inhibitor
cocktail [~3× the amount typically used]).
A1R and P2Y2R immunoreactivity intensities were much
stronger than that observed in the previous condition when used
fewer brains (Fig. 2a, b, f). The immunoblotting signal intensities
of coprecipitated P2Y2R and anti-A1R were also stronger
(Fig. 2d). The intensities of the immunoblot bands after
co-immunoprecipitation with the new methods were significantly
higher than those in the previous conditions (Fig. 3). These results
indicated that the band intensity was significantly greater,
suggesting the effectiveness of the improved conditions used in the
new method.
4 Notes
All steps of the experiments should be performed on ice or in

a cold room (<4 °C) to inhibit the catalytic activation of pro-
tease. Additionally, freezing the samples should be avoided. In
Fig. 3 Signal intensities of immunoprecipitated anti-A1R analyzed by immunoblotting with anti-P2Y2R and
ImageJ. Numbers 1, 2, 3, and 4 represent band intensities of A1R co-immunoprecipitated from the cerebral
cortex, cerebellum, hippocampus, and caudate putamen, respectively (Figs. 1d and 2d). The black and gray
columns indicate the results using the old method vs. the improved method, respectively. The lowest threshold
level for calculation by ImageJ was fixed using the weakest band (2; black column). Intensity values were
compared using Student’s t-tests. *: P < 0.01
co-immunoprecipitation experiments, denaturation of molecules

caused by freezing markedly decreases the binding ability of
GPCRs. For co-immunoprecipitation of A1R and P2Y2R, exsan-
guination was critical to obtain a sufficient co-immunoprecipitation
signal; the presence of blood in the brain tissue seemed to be
detrimental to the co-immunoprecipitation (Fig. 2a′, d′).
5 Conclusions
We successfully demonstrated A1R and P2Y2R hetero-

oligomerization in brain tissue [14] using a robust co-
immunoprecipitation procedure based on our previous study in
transfected HEK293T cells [12]. If molecular interaction is
observed in co-immunoprecipitation using cultured cells, similar
findings are possible in tissue. However, other reports many have
omitted the tissue findings due to the low Western blot signal
intensity after co-immunoprecipitation. Ex vivo and in vivo evi-
dence of molecular interactions is critical for evaluation during the
drug development process, and GPCRs are a drug discovery target
[17–19]. It may be necessary to investigate hetero-oligomeric in
addition to monomeric states using co-immunoprecipitation meth-
ods during drug discovery targeting GPCRs, as G protein-
associated signal intensities change when in the hetero-oligomeric
state [20]. The effects of a new drug targeting GPCRs may vary
due to cross talk or hetero-oligomerizations of GPCRs. Therefore,
GPCR cross talk data supported by co-immunoprecipitation
in vivo at an early stage are likely very important during drug
discovery.
In conclusion, we presented optimized GPCR co-
immunoprecipitation methods to identify GPCR oligomerization,
which may alter the signal intensity. Empirical data on GPCR inter-
actions are necessary to accurately evaluate the effects of drugs
targeting GPCRs in clinical trials and drug discovery studies.

Therefore, we strongly recommend detection of heteromeric
GPCR oligomerization (or any other molecular interaction) using
co-immunoprecipitation.
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Chapter 2
Co-immunoprecipitation Methods to Identify Associated

Proteins with Estrogen Receptor α at Postsynaptic Density
in Brain Tissue
Gen Murakami and Suguru Kawato
Abstract
Co-immunoprecipitation is a traditional method but still one of the most powerful tools to identify novel
interaction partners by increasing its specificity and decreasing false-positive result. In the current work, we
show a robust procedure where the use of two sequential approaches (separation of postsynaptic density
by centrifuge separation method in combination with detergent treatment and the use of specific antibod-
ies against target proteins for its precipitation) improved the specificity of co-immunoprecipitation meth-
ods. Mass spectrometric analysis also helped to identify novel-associated proteins with target proteins
without any bias of suspected interaction partners.
Key words Co-immunoprecipitation, Centrifuge separation, Brain, Estrogen receptor, Postsynaptic

density, Chromatin immunoprecipitation, Major histocompatibility complex
1 Introduction
1.1 Co-immuno- Information of protein-protein interactions is very important to

precipitation: investigate the molecular mechanisms which underlie various phys-
A Powerful Tool iological events. To identify associated proteins with targeted pro-
for Protein-Protein teins, several novel methods have been reported, and some
Interactions traditional methods have been improved over decades [1]. Among
these methods, co-immunoprecipitation, one of the most tradi-
tional methods, is still one of the most powerful tools and the most
often used for this purpose [2]. This is probably due to its high
sensitivity and relatively reliable detection of associated proteins
using specific antibodies under native conditions without overex-
pressions of target proteins and compatibility with mass spectro-
metric identification of proteins. Chromatin immunoprecipitation
(ChIP) is also a method of co-immunoprecipitation to identify
associated DNA fragments with target proteins and offers high
sensitivity and reliable detection of associated DNA fragments
Yuji Odagaki and Dasiel O. Borroto-Escuela (eds.), Co-Immunoprecipitation Methods for Brain Tissue, Neuromethods, vol. 144,
https://doi.org/10.1007/978-1-4939-8985-0_2, © Springer Science+Business Media, LLC, part of Springer Nature 2019
9
10 Gen Murakami and Suguru Kawato
using polymerase chain reaction. This method has also given us a

lot of information about mechanisms underlying physiological
events [3].
1.2 Limitation On the other hand, co-immunoprecipitation has some limitations.

of Co-immuno- One of the limitations is that this method cannot distinguish direct
precipitation and indirect interactions. Thus, identified associated proteins by
this method can interact directly with target proteins or indirectly
with target proteins as one of the proteins in the complex includ-
ing the target proteins. Another difficulty is that this method often
leads to false-positive results, although this possibility is considered
to be much less than other methods such as yeast two-hybrid
method [2]. This problem is mainly originated from non-specific
binding of antibodies which is used for precipitation of target pro-
teins. This tendency is much more evident when using tissue sam-
ples than cell culture samples. This is probably because tissue
samples include various types of cells that do not express target
proteins. Particularly in brain tissues, only 10% of cells are neurons,
and the other 90% of cells are glial cells. Therefore, the portion of
target proteins particularly expressed in specific neurons is consid-
ered to be much smaller. This false-positive result occurs more
often when target proteins are expressed less than other proteins
despite its important roles in physiological events. As we will see
later, estrogen receptor alpha (ERα), famous for regulating repro-
ductive functions in reproductive organs, is a good example because
expression level of ERα in the hippocampus is lower than one tenth
of ERα expressed in the ovary [4]. However, decades of studies
have demonstrated that ERα in the hippocampus plays important
roles in the regulation of synaptic plasticity [4–6].
1.3 Improving An approach to overcome these problems is using antibodies which

Specificity show good specificity for the target proteins. Although antibodies
of Co-immuno- predominantly bind to the epitope within target proteins, other
precipitation non-specific proteins without the epitope can also interact with the
antibodies. Specificity of antibodies is decided by the ratio of bind-
ings to specific proteins to non-specific proteins, and antibodies
with low specificity tend to lead to false-positive results. This speci-
ficity also depends on the type of antibodies such as polyclonal or
monoclonal. Particularly, polyclonal antibodies tend to have large
variability of their specificities, and purification of antibodies is par-
ticularly important for improving their specificities. Polyclonal
antibodies with different lot numbers may also have different spec-
ificities. Therefore, it is important to evaluate the specificity of anti-
bodies before co-immunoprecipitation, particularly when using
brain tissues.
Another approach to overcome the problems is separation of
samples before co-immunoprecipitation. This pre-separation step
can easily remove non-specific proteins and increase the ratio of
Co-immunoprecipitation of Estrogen Receptor Alpha 11
antibody bindings to specific proteins to non-specific proteins in

the co-immunoprecipitation experiment. Centrifuge separation is
suitable for the step of pre-separation because most of proteins are
localized at a specific subcellular region such as the nucleus, cellu-
lar membrane, endoplasmic reticulum, and cytoplasm. Centrifuge
separation can easily separate these specific regions from others and
remove a large part of nontarget proteins. Furthermore, centrifuge
separation in combination with detergent treatment can separate
neuron-specific subregions such as the postsynaptic density (PSD)
or presynaptic vesicles which play important roles in the regulation
of synaptic transmissions and plasticity in the brain [7–9]. Some
proteins are localized at several subcellular regions at the same time
by associating with different proteins, having different roles in
physiological events. Centrifuge separation of a specific subcellular
region allows us to investigate target proteins in a particular region
by removing the other regions. Therefore, co-immunoprecipitation
with specific antibodies following centrifuge separation makes pos-
sible to easily and reliably detect associated proteins with target
proteins localized at a specific subcellular region.
After co-immunoprecipitation, associated proteins are gener-
ally detected by western blotting using specific antibodies against
suspected interaction partners with the target proteins. Although
antibodies detect associated proteins with high sensitivity, this
property may also lead to false-positive results of detecting a small
amount of contaminated proteins. To reduce this possibility, it is
general to reverse co-immunoprecipitation where antibodies
against detected interaction partners are used for co-
immunoprecipitation and antibodies against original target pro-
teins are used for the detection in western blotting. However,
western blotting detection cannot discover novel interaction part-
ners. Mass spectrometric analysis is the most powerful method to
identify associated proteins with high sensitivity without any bias
of suspected interaction partners.
In this chapter, we show how these approaches improve the
specificity of co-immunoprecipitation by introducing a study that
identified associated proteins with estrogen receptor α (ERα) local-
ized at the PSD in rat brain. ERα is a nuclear receptor and localized
in the cytoplasm in the absence of its ligand, 17β-estradiol. By
binding with estradiol, ERα is transferred into nuclei and binds
with estrogen response element to regulate gene expressions under
the element [10]. In addition to this slow/genomic (within days)
functions, it has been found that ERα is also localized at the PSD,
and its binding with estradiol rapidly (within 2 h) modulates syn-
aptic plasticity [4–6]. Although it is considered that ERα is local-
ized at the PSD by anchoring to its scaffold proteins, these
associated proteins have not been identified. Using co-
immunoprecipitation with specific antibodies and centrifuge sepa-
ration method in combination with a detergent treatment, we
successfully purified ERα localized at the PSD and identified its

associated proteins by mass spectrometry.
2 Materials and Methods
2.1 Evaluation Before co-immunoprecipitation, our group assessed specificity

of Antibody Specificity of six antibodies against ERα by western blotting using the rat
Against Estrogen hippocampus as a brain tissue sample. The ovary was also used
Receptor α as a positive control. These antibodies included homemade
polyclonal antibody RC-19 which was column-purified with
antigens of rat and mouse ERα C-terminal 19 amino acids
(C-HSLQTYYIPPEAEGFPNTI) [4]. Other antibodies were com-
monly used or commercially available antibodies. MC-20 is poly-
clonal antiserum against C-terminal 20 amino acids of rat and
mouse ERα [11]. Using RC-19, we observed only a single protein
band at 67 kDa of ERα molecular weight in the hippocampus
homogenate and a very strong band at the same molecular weight
in the ovary homogenate (Fig. 1a). We also confirmed that this
67 kDa band was absent in the hippocampus of ERα knockout
mice [4, 5]. On the other hand, MC-20 is one of the most often
used antiserums to detect ERα and showed a band at 67 kDa in the
ovary homogenate. However, this 67 kDa band was not detectable
in the hippocampus sample, and major bands were observed at dif-
ferent molecular weights. MC-20 also showed these non-specific
bands in the hippocampus of ERα knockout mice [4]. This ten-
dency also can be observed in immunohistochemistry [4]. The
staining of RC-19 can be observed in the hippocampus of wild-
type mice but not in ERα knockout mice. On the other hand,
MC-20 staining was observed in both genotypes. These results
show that the use of antibodies purified with antigens from antise-
rum is important to increase the specificity, particularly in tissues
where target protein expression is low. Therefore, RC-19 is
expected to have high specificity for ERα in the co-
immunoprecipitation experiment. It is important to know that the
specificity of antibodies cannot be necessarily the same between
different experiments such as immunoprecipitation, western blot-
ting, and immunohistochemistry. However, evaluation of antibody
specificity in an experiment can still give important information of
the specificity in another experiment.
2.2 Centrifuge There are various choices of methods which can be used for purifi-
Separation cation of proteins before co-immunoprecipitation. Centrifuge sep-
of Postsynaptic aration is a traditional method to separate subcellular regions and
Density suitable for co-immunoprecipitation. In general centrifuge separa-
tion protocol, tissue samples can be separated to the nuclear frac-
tion (P1), plasma membrane fraction (P2), microsomal fraction
(P3), and cytoplasmic fraction (S3). In brain tissues, the P2 fraction
A
WB: RC-19 AS409 C1355 SRA1010 MC-20 HC-20
67 kDa
(ERα)
B WB : RC-19 C WB : WB :
Gsα VAMP-2
Synapto-
Ras physin
67 kDa Flotillin-1
(ERα) IP3R
Synapto- Spinophilin
physin PSD-95
ERα ERα
D
WB : RC-19
ERα
Fig. 1 Western blotting analysis using rat brains. (a) Western blotting of ERα with six antibodies to evaluate
their specificities. The hippocampus and ovaries diluted to one third of hippocampal proteins were used as a
brain sample and positive control, respectively. (b) Western blotting of ERα with RC-19 in some brain regions
such as the cerebral cortex, hippocampus, hypothalamus, and cerebellum. The ovary sample diluted to 1/20
of proteins in other brain regions was also used as a positive control. (c) Western blotting of several region-
specific markers in fractions prepared by centrifuge separation such as high-density membrane (HDM), low-
density membrane (LDM), postsynaptic density (PSD), raft, triton-soluble synaptosome, and synaptosome. (d)
Western blotting of ERα with RC-19 in fractions prepared by centrifuge separation such as the nucleus, LDM,
cytoplasm, and PSD. The ovary sample was also used as a positive control
can be further separated to the myelin, synaptosomal, and mito-

chondrial fractions [9]. The postsynaptic density (PSD) fraction
can be separated from the synaptosomal fraction by centrifuge
separation in combination with detergent treatment [7, 8].
Our group has applied this method to separate ERα localized
at the PSD (Fig. 2a). Two male Wistar rats aged 12 weeks (Saitama
Experimental Animal Supply, Saitama, Japan) were deeply anesthe-
tized and decapitated. Then, the whole brains were quickly
removed and placed in ice-cold homogenization buffer, containing
0.32 M sucrose, 5 mM HEPES, 25 mM KCl, 4 mM EDTA-4Na,
1 mM Na3VO4, 50 mM NaF, and protease inhibitor cocktails
(Roche, Laval, QC, Canada), and pH was adjusted to 7.3. Because
synaptosomes are caused to coacervate by high concentrations of
univalent ions or low concentrations of divalent ions, it is impor-
tant to optimize the concentration of these ions in the homogeni-
zation buffer [9]. We used whole brains because the localization of
ERα at the PSD and its rapid modulation of synaptic plasticity have
been shown in various brain regions. However, we removed cere-
bellums because this region contains much less ERα than other
brain regions (Fig. 1b). The brain sample was homogenized (10%
wt/vol) in homogenization buffer and centrifuged at 830 × g for
10 min. The pellet was used as P1, although this fraction contains
cell debris in this protocol. Supernatant from the first centrifuga-
tion was subjected to centrifugation at 10,000 × g for 20 min, and
the pellet (P2) was retrieved.
The P2 fraction contains the synaptosomal fraction, which con-
sists of synaptic terminals pinched-off from neurons by homogeni-
zation, and pre- and postsynapses are included in this fraction [9].
The synaptosomal fraction is generally prepared by subsequent
ultracentrifuge separation of the P2 fraction using 0.85/1.0/1.2 M
sucrose step gradient and collected from the interface between
1.0 M and 1.2 M sucrose. Although relatively pure PSD fraction is
separated from this fraction (Fig. 1c), we skipped the step of syn-
aptosomal separation to avoid loss of target proteins. The PSD
fraction is separated by its characteristic biochemical properties of
detergent resistance [7, 8]. Because the PSD consists of tightly
packed proteins important for synaptic transmissions such as recep-
tors for neurotransmitters, cell adhesion proteins, scaffold proteins,
and signaling molecules [12], the PSD complexes cannot be dis-
sociated by mild detergents such as Triton X-100. Thus, P2 was
solubilized in 40 mL homogenization buffer without sucrose con-
taining 0.5% Triton X-100 for 30 min at 4 °C using a stirrer, and
the triton-insoluble P2 pellet fraction was collected by centrifuga-
tion at 15,000 × g for 30 min and used as PSD-rich samples for
co-immunoprecipitation. Further centrifugation of the superna-
tant obtained from the fractionation of P2 at 25,000 × g for 60 min
gave the high-density membrane fraction. The resultant superna-
tant was centrifuged again at 100,000 × g for 60 min, and the
A
Brain tissues
830gʹ10 min
sup: S1
10,000gʹ20 min
ppt: P1 (Nucleus)
sup: S2
25,000gʹ1 hr
Solubilization with Triton X-100 ppt: P2
for 30 min at 4Υ
sup: S3
15,000gʹ30 min 100,000gʹ1 hr
ppt: HDM
Trioton-soluble P2: sup sup: S4 (Cytoplasm)
Trioton-insoluble P2: ppt sup: LDM
B
Cytoplasm (16%)
HDM + LDM (2%)

Triton-insoluble P2 (1%)
Triton-soluble P2 (4%)
P1 (76%)
Fig. 2 Centrifuge separation of triton-insoluble P2 fraction. (a) Schematic diagram of centrifuge separation.
(b) Proportion of protein amounts in fractions prepared by centrifuge separation. P1 (nucleus), triton-soluble
P2, triton-insoluble P2, P3 (membrane), and S3 (cytoplasm) account for 76%, 4%, 1%, 2%, and 16% of total
proteins, respectively
ellet and the supernatant were collected separately as the low-

p
density synaptic membrane fraction (LDM) and cytoplasm frac-
tion, respectively [4]. The LDM faction contained proteins
specifically localized at synaptic vesicles (Fig. 1c). Western blotting
analysis showed that ERα was localized at the PSD fraction as well
as nucleus, cytoplasm, and LDM fractions (Fig. 1d). This centri-
fuge separation removed ERα in subregions other than the PSD
and allows us to analyze the associated proteins with PSD-localized
ERα. It is important that, the triton-insoluble P2 fraction contained

less than 1% of total proteins in the brain homogenate (Fig. 2b).
Therefore, this single step of centrifuge separation greatly reduced
nontarget proteins and is expected to increase the specificity of co-
immunoprecipitation more than 100-folds.
2.3 Co-immuno- Although solubilization of samples is necessary before co-

precipitation immunoprecipitation, the use of strong detergents or high concen-
of Estrogen Receptor tration of detergents interferes protein-protein interactions. Thus,
α-Associated Proteins it is important to optimize the combination of detergents and their
concentrations to solubilize samples but not to interfere the asso-
ciation of target proteins. As we used in this experiment, low con-
centration of sodium dodecylsulfate (SDS) combined with mild
detergents of sodium deoxycholate and NP-40 is often used for
solubilization of the PSD [7, 8]. We solubilized triton-insoluble P2
pellet using a stirrer for 1 h at 4 °C in 3 mL immunoprecipitation
buffer containing 0.1% SDS, 1% sodium deoxycholate, 1% NP-40,
5 mM HEPES, 25 mM KCl, 4 mM EDTA-4Na, 1 mM Na3VO4,
50 mM NaF, and protease inhibitor cocktails (Roche), and pH was
adjusted to 8.1.
For precipitation of target proteins, we need to select beads to
link with antibodies, such as agarose, sepharose, or magnetic beads
linked to protein G, protein A, or secondary antibodies. Our group
compared these beads and selected magnetic beads linked to anti-
rabbit IgG (Dynabeads M-280 anti-rabbit IgG; Thermo Fisher
Scientific Inc., Waltham, MA, USA) for the following reasons.
First, some of these beads non-selectively associate with proteins in
samples and possibly lead to false-positive results. Anti-rabbit IgG-
linked magnetic beads showed less associations with non-specific
proteins than other beads. Second, centrifugation is necessary to
collect agarose and sepharose beads in co-immunoprecipitation.
However, centrifugation also non-specifically precipitates insoluble
protein complexes. Precipitation of insoluble protein complexes
was also observed even when these insoluble complexes were
removed by several centrifugations of triton-insoluble P2 samples
before co-immunoprecipitation. The use of magnetic beads solved
this problem because these beads can be collected on the wall of
sample tubes by a magnet attached to the outside of the wall with-
out centrifugation.
Our group reacted 6.7 × 107 of magnetic beads linked to anti-
rabbit IgG with 10.0 μg of RC-19, a homemade rabbit polyclonal
antibody against ERα, in phosphate buffer saline containing 0.1%
bovine serum albumin by rotating sample tubes for 3 h at
4 °C. Then, RC-19 was covalently coupled with anti-rabbit IgG by
dimethyl pimelimidate (Pierce, Rockford, IL, USA) to minimize
the contamination of RC-19 in the elution process of target pro-
teins. After washing with immunoprecipitation buffer, the mag-
netic beads linked with RC-19 were reacted with 1.5 mL
triton-insoluble P2 sample by rotating sample tubes overnight at

4 °C. It is important to prepare a negative control using normal
rabbit serum (NRS) exactly the same as RC-19 for the identifica-
tion of non-specifically associated proteins. For separation of SDS-
PAGE, ERα was eluted by sample buffer containing 125 mM
Tris-HCl (pH 6.8), 5 mM 2-mercaptoethanol, 10% sucrose, 6%
sodium dodecylsulfate (SDS), and 0.002% bromophenol blue by
incubating for 5 min at 100 °C, followed by further elution by
adding dithiothreitol (100 mM) and incubating for 10 min at
100 °C. After protein separation by SDS-PAGE, these gels were
stained overnight with 0.1% silver using the silver stain MS kit
(Wako, Tokyo, Japan).
In the silver staining analysis, many protein bands were
observed in precipitated samples not only with RC-19 but also
NRS at the same molecular weight (Fig. 3). This result shows that
many proteins were non-specifically precipitated with antibody-
linked magnetic beads, while we increased specificity of co-
immunoprecipitation in various steps. However, some specific
bands were observed particularly in RC-19 but not in NRS sam-
ples, and these bands were considered ERα-associated proteins. We
also precipitated and silver-stained ERα-associated proteins in the
LDM fraction. We can see that the patterns of specific bands pre-
cipitated with RC-19 were quite different between the triton-
insoluble P2 and LDM samples. This difference indicates that ERα
is localized in the different subcellular regions associating with dif-
ferent proteins.
2.4 Mass For mass spectrometric identification of ERα-associated proteins,

Spectrometric RC-19-specific bands were isolated from the gels using razor
Analysis of ERα- blades. These gel fragments were destained with the destaining
Associated Proteins solution of the silver stain kit, and the proteins were digested in-gel
with trypsin (V5280; Promega, Madison, WI, USA). Digested
peptides were extracted from gel pieces with 50 μL 50% acetoni-
trile and 5% trifluoroacetic acid. ZipTip (Millipore, Billerica, MA,
USA) was used for purification of peptides. A matrix-assisted laser
desorption ionization (MALDI) time-of-flight (TOF) mass spec-
trometer (Shimazu, Tokyo, Japan) was used for detection of ERα-
associated proteins. Samples were prepared by mixing peptide
solution with 0.3 μL matrix solution on a MALDI plate. All masses
are reported as monoisotopic mass values. Peptides were identified
using the Mascot search program (http://www.matrixscience.
com) to perform theoretical trypsin digests and search for potential
unmodified tryptic peptides.
Many tryptic peptides prepared from gel fragments of 67 kDa
in the triton-insoluble P2 and LDM samples were attributed to the
tryptic peptides from ERα the same as our previous report [4]. We
also identified precipitation of ERα by western blotting using
RC-19 (Fig. 3b). When antibodies raised from the same animal
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THROUGH KIKUYU TO
GALLA-LAND
INTRODUCTION.
My friend, George Henry West, and myself left Cairo in the latter part
of the year 1899, with the intention of proceeding to Uganda viâ
Zanzibar and Mombasa. George was an engineer in the service of
the Irrigation Department of the Egyptian Government, and had
gained a large and varied experience on the new works on the
Barrage below Cairo, then being concluded, and in building, running,
and repairing both locomotives and launches. As a profession I had
followed the sea for three years, leaving it in 1896 in order to join the
British South African Police, then engaged in subduing the native
rebellion in Mashonaland. At the conclusion of hostilities I wandered
over South Africa, and finally found my way to Egypt, where I met
George West. A year later, accompanied by George, I was on my
way southwards again, en route for British East Africa.
When George and I left Cairo, our idea was to go up-country as far
as the Lake Victoria Nyanza, as we considered it extremely probable
that there would be something for us to do in the engineering line,
either in building launches or in the construction of small harbour
works.
We reached Mombasa in due course, and from there proceeded to
Nairobi by the railway then in course of construction to Uganda.
Nairobi is 327 miles from the coast, and is an important centre, being
the headquarters of both the Civil Administration of the Protectorate
and the Uganda railway. On our arrival, George received an offer,
which he accepted, to go up to the lake with a steamer, which was
then on the way out from England in sections, and on his arrival at
the lake with it to rebuild it. I remained in Nairobi.
In course of time I met the personage referred to in these pages
as “El Hakim,”[1] whom I had known previously by repute. He was
said to be one of the most daring and resolute, and at the same time
one of the most unassuming Englishmen in the Protectorate; a dead
shot, and a charming companion. He had been shooting in
Somaliland and the neighbourhood of Lake Rudolph for the previous
four years, and many were the stories told of his prowess among
elephant and other big game.
It was with sincere pleasure, therefore, that I found I was able to
do him sundry small services, and we soon became fast friends. In
appearance he was nothing out of the common. He was by no
means a big man—rather the reverse, in fact—and it was only on
closer acquaintance that his striking personality impressed one.
He had dark hair and eyes, and an aquiline nose. He was a man
of many and varied attainments. Primarily a member of the medical
profession, his opinions on most other subjects were listened to with
respect. A very precise speaker, he had a clear and impartial manner
of reviewing anything under discussion which never failed to impress
his hearers.
He was a leader one would have willingly followed to the end of
the earth. When, therefore, he proposed that I should accompany
him on an ivory trading expedition to Galla-land, that vast stretch of
country lying between Mount Kenia on the south and Southern
Somaliland on the north, which is nominally under the sphere of
influence of the British East African Protectorate, I jumped at the
chance; and it was so arranged. He had been over much of the
ground we intended covering, and knew the country, so that it
promised to be a most interesting trip.
About this time I heard from George that he was coming down
country, as the steamer parts had not all arrived from England, and
consequently it would probably be months before it would be ready
for building. He had also had a bad attack of malarial fever in the
unhealthy district immediately surrounding the lake at Ugowe Bay,
and altogether he was not very fit. I suggested to El Hakim that
George should join us in our proposed expedition, to which he
readily agreed; so I wrote to George to that effect.
To render the prospect still more inviting, there existed a certain
element of mystery with regard to the river Waso Nyiro (pronounced
Wasso Nēro). It has always been supposed to rise in the Aberdare
Range, but, as I shall show, I have very good reason to believe that it
rises in the western slopes of Kenia Mountain itself. The Waso Nyiro
does not empty itself into the sea, but ends in a swamp called
Lorian, the position of which was supposed to have been fixed by an
exploring party in 1893. But, as I shall also show in the course of this
narrative, the position of Lorian varies.
The upper reaches of the Waso Nyiro were visited by the explorer
Joseph Thompson, F.R.G.S., on his way to Lake Baringo during his
memorable journey through Masai Land in 1885.
In 1887-1888 a Hungarian nobleman, Count Samuel Teleki von
Czeck, accompanied by Lieutenant Ludwig von Hohnel, of the
Imperial Austrian Navy, undertook the stupendous journey which
resulted in the discovery of Lakes Rudolph and Stephanie. Count
Teleki, on his journey north, crossed the Waso Nyiro at a point in
North-West Kenia near its source, while Lieutenant von Hohnel went
two or three days’ march still further down-stream.
A few years later, in 1892-1893, Professor J. W. Gregory, D.Sc., of
the Natural History Museum, South Kensington, made, single-
handed, a remarkable journey to Lake Baringo and Mount Kenia,
and in the teeth of almost insuperable difficulties, ascended the
western face of that mountain and climbed the peak.
At the same time, in the latter part of 1892, an American, Mr.
William Astor Chanler, accompanied by Count Teleki’s companion
and chronicler, Lieutenant von Hohnel, started from a point in
Formosa Bay on the East Coast, and made his way along the course
of Tana River to North-East Kenia, intending later to go on to Lake
Rudolph, and thence northward. He and his companion, deceived by
the reports of the natives, which led them to believe that the Waso
Nyiro emptied itself into an extensive lake, and fired by the idea of
the possible discovery of another great African lake, made their way
down to the Waso Nyiro, and after a fearful march, enduring the
greatest hardships, eventually reached Lorian. To their great
disappointment, it proved to be nothing more than a swamp, and
they turned back without examining it. A few weeks later, Lieutenant
von Hohnel, having been seriously injured by a rhinoceros, was sent
down to the coast, his life being despaired of. Shortly afterwards Mr.
Chanler’s men deserted him in a body, and returned to the coast
also, thus bringing his journey to a premature conclusion; a much-to-
be-regretted ending to a well-planned and well-equipped expedition.
As Mr. Chanler was returning to the coast he met Mr. A. H.
Neumann coming up. Mr. Neumann spent the greater part of 1893 in
shooting elephants in the Loroghi Mountains, after going north to
Lake Rudolph. He also crossed the Waso Nyiro at a point north-east
of Mount Kenia.
During the time Mr. Neumann was shooting in the Loroghi
Mountains he was obliged to make periodical visits to M’thara, in
North-East Kenia, in order to buy food from the natives, and on one
such excursion he met Dr. Kolb, a German scientist, who was
exploring North Kenia.
Dr. Kolb ascended Mount Kenia from the north, and then returned
to Europe. An interesting account of his ascent of the mountain is
published in Dr. Petermann’s “Mitteilungen” (42 Band, 1896). Dr.
Kolb then returned to Kenia in order to continue his observations, but
he was unfortunately killed by a rhinoceros a couple of marches
north of M’thara.
Lorian, therefore, with the exception of Mr. Chanler’s hurried visit,
was practically unexplored. At the commencement of our trip, El
Hakim proposed that, if an opportunity occurred of visiting Lorian, we
should take advantage of it, and endeavour to supplement Mr.
Chanler’s information. As will be seen, an opportunity did present
itself, with what result a perusal of this account of our expedition will
disclose.
FOOTNOTES:
[1] Anglice, “The Doctor.”
CHAPTER I.
PREPARATIONS AND START.
Engaging porters—Characteristics of Swahili, Wa’Nyamwezi, and

Wa’Kamba porters—Selecting trade goods—Provisions—Arms
and ammunition—The Munipara—Sketch of some principal
porters—Personal servants—List of trade goods taken—
Distributing the loads—Refusal of the Government to register our
porters—Reported hostility of the natives—Finley and Gibbons’
disaster—Start of the Somali safaris—We move to Kriger’s Farm
—I fall into a game-pit—Camp near Kriger’s Farm—Visitors—The
start.
One of the most important items in the organization of a “safari”
(caravan) is the judicious selection of the men. Choosing ours was a
task that gave us much trouble and vexation of spirit. El Hakim said
that for all-round usefulness the Wa’kamba were hard to beat, and
thought that we had better form the bulk of the safari from them, and
stiffen it with a backbone of Swahilis and Wa’Nyamwezi, as, though
the Wa’kamba were very good men when well handled, in the
unlikely event of hostilities with the natives it would be advisable to
strengthen them with an addition from the lustier tribes. To that end
we proposed to engage a dozen Swahili and half that number of
Wa’Nyamwezi. Porters at that time were very scarce; but having
secured one or two good men as a nucleus, we sent them into the
bazaar at Nairobi to bring us any other men they could find who
wanted employment.
The Swahilis are natives of Zanzibar and the adjacent coasts.
They are of mixed—very mixed—descent, being mainly the offspring
of various native slaves and their Arab masters. They were originally
a race of slaves, but since the abolition of slavery they have become
more and more independent, and they now consider themselves a
very superior race indeed. They call themselves “Wangwana”
(freemen), and allude to all other natives as “Washenzi” (savages).
They are incorrigibly conceited, and at times very vicious, lazy,
disobedient, and insolent. But once you have, by a judicious display
of firmness, gained their respect, they, with of course some
exceptions, prove to be a hardy, cheerful, and intelligent people,
capable of enduring great hardships without a too ostentatious
display of ill-feeling, and will even go so far as to make bad puns in
the vernacular upon their empty stomachs, the latter an occurrence
not at all infrequent in safari work away from the main roads.
The Wa’kamba, on the whole, are a very cheerful tribe, and though
of small physique, possess wonderful powers of endurance, the
women equally with the men. We calculated that some of our men, in
addition to their 60-lb. load, carried another 30 lbs. weight in
personal effects, rifle, and ammunition; so that altogether they
carried 90 lbs. dead weight during one or sometimes two marches a
day for weeks at a stretch, often on insufficient food, and sometimes
on no food at all.
The Wa’Nyamwezi are, in my opinion, really more reliable than
either the Swahili or Wa’kamba. They come from U’Nyamwezi, the
country south and east of Lake Victoria Nyanza. We had six of them
with us, and we always found them steady and willing, good porters,
and less trouble than any other men in the safari. They were very
clannish, keeping very much to themselves, but were quiet and
orderly, and seldom complained; and if at any time they imagined
they had some cause for complaint, they formed a deputation and
quietly stated their case, and on receiving a reply as quietly returned
to their fire—very different from the noisy, argumentative Swahili.
They appear to me to possess the virtues of both the Swahilis and
Wa’kamba without their vices. The Wa’kamba’s great weakness
when on the march was a penchant for stealing from the native
villages whatever they could lay their hands on, being encouraged
thereto by the brave and noble Swahilis, who, while not wishing to
risk our displeasure by openly doing likewise, urged on the simple
Wa’kamba, afterwards appropriating the lion’s share of the spoil: that
is, if we did not hear of the occurrence and confiscate the spoil
ourselves.
We had pitched our tent just outside the town of Nairobi, and
proceeded to get together our loads of camp equipment, trade
goods, and provisions: no easy task on an expedition such as ours,
where the number of carriers was to be strictly limited.
In the first place, we required cloth, brass wire, iron wire, and
various beads, in sufficient quantities to buy food for the safari for at
least six months. Provisions were also a troublesome item, as,
although we expected to live a great deal upon native food, we
required such things as tea, coffee, sugar, jam, condiments, and also
medicines. The question was not what to take, but what not to take.
However, after a great amount of discussion, lasting over several
days, we settled the food question more or less satisfactorily.
During this time our recruiting officers were bringing into camp
numbers of men who, they said, wanted to take service with us as
porters. Judging from the specimens submitted for our approval, they
seemed to have raked out the halt, the lame, and the blind. After
much trouble we selected those whom we thought likely to be
suitable, and gave them an advance of a few rupees as a retaining
fee, with which, after the manner of their kind, they immediately
repaired to the bazaar for a last long orgie.
There was also the important question of arms and ammunition to
be considered, as, although we did not expect any fighting, it would
have been foolish in the extreme to have entered such districts as
we intended visiting without adequate means of self-defence. We
concluded the twenty-five Snider rifles used by El Hakim on a
previous trip would suffice. Unfortunately, we could get very little
ammunition for them, as at that time Snider ammunition was very
scarce in Nairobi, one reason being that it had been bought very
largely by a big Somali caravan under Jamah Mahomet and Ismail
Robli, which set out just before us, bound for the same districts.
We, however, eventually procured five or six hundred rounds: a
ridiculously inadequate amount considering the distance we were to
travel and the time we expected to be away.
With regard to our armament, El Hakim possessed by far the best
battery. His weapons consisted of an 8-bore Paradox, a ·577
Express, and a single-barrelled ·450 Express, all by Holland and
Holland. The 8-bore we never used, as the ·577 Express did all that
was required perfectly satisfactorily. The 8-bore would have been a
magnificent weapon for camp defence when loaded with slugs, but
fortunately our camp was never directly attacked, and consequently
the necessity for using it never arose. The ·557 was the best all-
round weapon for big game such as elephant, rhinoceros, and
buffalo, and never failed to do its work cleanly and perfectly. Its only
disadvantage was that it burnt black powder, and consequently I
should be inclined, if I ever made another expedition, to give the
preference to one of the new ·450 or ·500 Expresses burning
smokeless powder, though, as I have not handled one of the latter, I
cannot speak with certainty. El Hakim’s ·450 Express was really a
wonderful weapon, though open to the same objection as the ·557—
that of burning black powder. It was certainly one of the best all-
round weapons I ever saw for bringing down soft-skinned game. It
was a single-barrelled, top-lever, hammer-gun, with flat top rib. The
sights were set very low down on the rib, to my mind a great
advantage, as it seems to me to minimize the chances of accidental
canting. Its penetrative power, with hardened lead bullets, was
surprising. I have seen it drop a rhinoceros with a bullet through the
brain, and yet the same projectile would kill small antelope like
Grant’s or Waller’s gazelles without mangling them or going right
through and tearing a great hole in its egress, thereby spoiling the
skin, which is the great cause of complaint against the ·303 when
expanding bullets are used.
I myself carried a ·303 built by Rigby, a really magnificent weapon.
I took with me a quantity of every make of ·303 expanding bullets,
from copper-tubed to Jeffry’s splits. After repeated trials I found that
the Dum-Dum gave the most satisfactory results, “since when I have
used no other.”
I also carried a supply of ·303 solid bullets, both for elephants and
for possible defensive operations. For rhinoceros, buffalo, or giraffe, I
carried an ordinary Martini-Henry military rifle, which answered the
purpose admirably. A 20-bore shot-gun, which proved useful in
securing guinea-fowl, etc., for the pot, completed my battery. George
carried a ·303 military rifle and a Martini-Henry carbine.
It was essential that we should have a good “Munipara” (head-
man), and the individual we engaged to fill that important position
was highly recommended to us as a man of energy and resource.
His name was Jumbi ben Aloukeri. Jumbi was of medium height,
with an honest, good-natured face. He possessed an unlimited
capacity for work, but we discovered, too late, that he possessed no
real control over the men, which fact afterwards caused us endless
trouble and annoyance. He was too easy with them, and made the
great mistake—for a head-man—of himself doing anything we
wanted, instead of compelling his subordinates to do it, with the
result that he was often openly defied, necessitating vigorous
intervention on our part to uphold his authority. We usually alluded to
him as “the Nobleman,” that being the literal translation of his name.
Next on the list of our Swahili porters was Sadi ben Heri, who had
been up to North Kenia before with the late Dr. Kolb, who was killed
by a rhinoceros a couple of marches north of M’thara, Sadi was a
short, stoutly built, pugnacious little man, with a great deal to say
upon most things, especially those which did not concern him. He
was a good worker, but never seemed happy unless he was
grumbling; and as he had a certain amount of influence among the
men, they would grumble with him, to their great mutual satisfaction
but ultimate disadvantage. His pugnacious disposition and lax
morals soon got him into trouble, and he, together with some of his
especial cronies, was killed by natives, as will be related in its proper
sequence.
Hamisi ben Abdullah was a man of no marked peculiarities, except
a disposition to back up Sadi in any mischief. The same description
applies to Abdullah ben Asmani and Asmani ben Selim.
Coja ben Sowah was a short, thick-set man, so short as to be
almost a dwarf. He was one of the most cheery and willing of our
men, so much so that it was quite a pleasure to order him to do
anything—a pleasure, I fear, we appreciated more than he did. On
receiving an order he would run to execute it with a cheery “Ay
wallah, bwana” (“Please God, master”), that did one good to hear.
Resarse ben Shokar was our “Kiongozi,” i.e. the leading porter,
who sets the step on the march and carries the flag of the safari. He,
also, always ran on receiving an order—ran out of sight, in fact; then,
when beyond our ken, compelled a weaker man than himself to do
what was wanted. I could never cure him of the habit of sleeping on
sentry duty, though many a time I have chased him with a stirrup-
strap, or a camp-stool, or anything handy when, while making
surprise inspections of the sentries, I had found him fast asleep. He
was valuable, however, in that he was the wit of the safari. He was a
perfect gas-bag, and often during and after a long and probably
waterless march we blessed him for causing the men to laugh by
some harmless waggish remark at our expense.
Sulieman was a big, hulking, sulky brute, who gave us a great deal
of trouble, and finally deserted near Lorian, forgetting to return his
rifle, and also absent-mindedly cutting open my bag and abstracting
a few small but necessary articles. Docere ben Ali, his chum, was
also of a slow and sullen disposition, though he was careful not to
exhibit it to us. When anything disturbed him he went forthwith and
took it out of the unfortunate Wa’kamba.
Of the Wa’kamba I do not remember the names except of two or
three who particularly impressed themselves on my memory. The
head M’kamba was known as Malwa. He was a cheerful, stupid idiot
who worked like a horse, though he never seemed to get any
“for’arder.” Another M’kamba, named Macow, afterwards succeeded
him in the headmanship of the Wa’kamba when Malwa was deposed
for some offence. We nicknamed Macow “Sherlock Holmes,” as he
seemed to spend most of his leisure hours prowling round the camp,
peering round corners with the true melodrama-detective-Hawkshaw
expression in his deep-set, thickly browed eyes. He would often
creep silently and mysteriously to our tent, and in a subdued whisper
communicate some trifling incident which had occurred on the
march; then, without waiting for a reply, steal as silently and
mysteriously away.
I must not conclude this chapter without some mention of our
personal servants. First and foremost was Ramathani, our head
cook and factotum. Ramathani had already been some three months
in my service as cook and personal servant, and a most capable
man I had found him. My acquaintance with him began one morning
when I had sent my cook, before breakfast, to the sokoni (native
bazaar) to buy bread, vegetables, etc. As he did not return I went
outside to the cook-house in some anxiety as to whether I should get
any breakfast. Several native servants were there, and they informed
me my cook was still in the bazaar, very drunk, and most likely would
not be back till noon. Of course, I was angry, and proceeded to show
it, when a soothing voice, speaking in very fair English, fell upon my
ear. Turning sharply, I was confronted by a stranger, a good-looking
native, neatly dressed in khaki.
“Shall I cook breakfast for master?” he inquired softly.
“Are you able?” said I.
“Yes, master.”
“Then do so,” I said; and went back to my quarters and waited with
as much patience as I could command under the circumstances.
In a quarter of an hour or so Ramathani—for it was indeed he—
brought in a temptingly well-cooked breakfast, such as I was almost
a stranger to, and at the same time hinted that he had permanently
attached me as his employer. My own cook turned up an hour or so
later, very drunk and very abusive, and he was incontinently fired
out, Ramathani being established in his stead.
Ramathani had two boys as assistants, Juma and Bilali. Juma was
an M’kamba. His upper teeth were filed to sharp points, forming most
useful weapons of offence, as we afterwards had occasion to notice.
Bilali was an M’Kikuyu, and a very willing boy. He was always very
nervous when in our presence, and used to tremble excessively
when laying the table for meals. When gently reproved for putting
dirty knives or cups on the table, he would grow quite ludicrous in his
hurried efforts to clean the articles mentioned, and would spit on
them and rub them with the hem of his dirty robe with a pathetic
eagerness to please that disarmed indignation and turned away
wrath.
Having finally secured our men, it only remained to pack up and
distribute the loads of equipment, provisions, trade goods, etc. We
did not take such a large quantity of trade goods as we should have
done in the ordinary course, as El Hakim already had a large
quantity in charge of a chief in North Kenia. The following is a list,
compiled from memory, of what we took with us:—
Unguo (Cloth).
2 loads Merikani (American sheeting).
2 ” kisuto (red and blue check cloths).
2 ” blanketi (blankets, coloured).
1 load various, including—
gumti (a coarse white cloth).
laissoes (coloured cloths worn by women).
kekois (coloured cloth worn by men).
Uzi Wa Madini (Wire).

seninge (iron wire, No. 6).
2 or 3 loads of masango (copper wire, No. 6).
masango n’eupe (brass wire, No. 6).
Ushanga (Beads).
sem Sem (small red Masai beads).
2 or 3 loads of sembaj (white Masai beads).
ukuta (large white opaque beads).
2 loads of mixed Venetian beads.
When all the loads were packed, they were placed in a line on the
ground; and falling the men in, we told off each to the load we
thought best suited to him. To the Swahilis, being good marching
men and not apt to straggle on the road, we apportioned our
personal equipment, tents, blankets, and table utensils. To the
Wa’Nyamwezi we entrusted the ammunition and provisions, and to
the Wa’kamba we gave the loads of wire, beads, cloth, etc. Having
settled this to our own satisfaction, we considered the matter settled,
and ordered each man to take up his load.
Then the trouble began. First one man would come to us and ask
if his load might be changed for “that other one,” while the man to
whom “that other one” had been given would object with much
excited gesticulation and forcible language to any alteration being
made, and would come to us to decide the case. We would then
arbitrate, though nine times out of ten they did not abide by our
decision. Other men’s loads were bulky, or awkward, or heavy, or
had something or other the matter with them which they wanted
rectified, so that in a short time we had forty men with forty
grievances clamouring for adjustment. We simplified matters by
referring every one to Jumbi, and having beaten an inglorious retreat
to our tents, solaced ourselves with something eatable till everything
was more or less amicably settled.
Nothing is more characteristic of the difference in the races than
the way in which they carry their loads. The Swahilis and
Wa’Nyamwezi, being used to the open main roads, carry their loads
boldly on their heads, or, in some cases, on their shoulders. The
Wa’kamba, on the other hand, in the narrow jungle paths of their
own district find it impossible, by reason of the overhanging
vegetation, to carry a load that way. They tie it up instead with a long
broad strip of hide, leaving a large loop, which is passed round the
forehead from behind, thus supporting the load, which rests in the
small of the back. When the strain on the neck becomes tiring they
lean forward, which affords considerable relief, by allowing the load
to rest still more upon the back. There were also six donkeys, the
property of El Hakim, and these were loaded up as well. A donkey
will carry 120 lbs., a weight equal to two men’s loads.
Finally, we had to register our porters at the Sub-Commissioner’s
office, as no safaris are allowed to proceed until that important
ceremony has been concluded, and the Government has pouched
the attendant fees. In our case, however, there appeared to be a
certain amount of difficulty. On delivering my application I was told to
wait for an answer, which I should receive in the course of the day. I
waited. In the afternoon a most important-looking official document
was brought to me by a Nubian orderly. In fear and trembling I
opened the envelope, and breathed a heartfelt sigh of relief when I
found that the Government had refused to register our porters, giving
as their reason that the districts we intended visiting were unsettled
and, in their opinion, unsafe, and therefore we should proceed only
at our own risk. We did not mind that, and we saved the registration
fee anyhow. The Government had already refused to register the
Somali’s porters, and they intimated, very rightly, that they could not
make any difference in our case.
Jamah Mahomet, who was in command of the Somali safari,
started off that day. He had with him Ismail Robli as second in
command. A smaller safari, under Noor Adam, had started a week
previously. Both these safaris intended visiting the same districts as
ourselves. We were fated to hear a great deal more of them before
the end of our trip.
In the evening I received a private note from one of the
Government officers, informing me that we were likely to have a
certain amount of trouble in getting across the river Thika-Thika
without fighting, as the natives of that district were very turbulent,
and advising us to go another way. My informant cited the case of
Messrs. Finlay and Gibbons by way of a cheerful moral.
Finlay and Gibbons were two Englishmen who had been trading
somewhere to the north of the Tana River. They had forty men or so,
and were trading for ivory with the A’kikuyu, when they were
suddenly and treacherously attacked and driven into their “boma”
(thorn stockade), and there besieged by quite six thousand natives.
From what I saw later, I can quite believe that their numbers were by
no means exaggerated. During a night attack, Finlay was speared
through the hand and again in the back, the wound in the back,
however, not proving dangerous. They managed to get a message
through to Nairobi, and some Nubian troops were sent to their relief,
which task they successfully accomplished, though only with the
greatest difficulty. It was not till six weeks after he received the
wound that Finlay was able to obtain medical assistance, and by that
time the tendons of his hand had united wrongly, so that it was
rendered permanently useless. This was a nice enlivening story,
calculated to encourage men who were setting out for the same
districts.
The following day I received a telegram from George to say that
he had arrived from Uganda at the Kedong Camp, at the foot of the
Kikuyu Escarpment, so I went up by rail to meet him. He looked very
thin and worn after his severe attack of fever. We returned to Nairobi
the same evening, and proceeded to our camp. El Hakim, who was
away when we arrived, turned up an hour later, and completed our
party. He had been to Kriger’s Farm about seven miles out. Messrs.
Kriger and Knapp were two American missionaries who had
established a mission station that distance out of Nairobi, towards
Doenyo Sabuk, or Chianjaw, as it is called by the Wa’kamba.
El Hakim, being anxious to get our men away from the pernicious
influence of the native bazaar, arranged that he would go on to
Kriger’s early on the following morning, and that George and I should
follow later in the day with the safari, and camp for the night near
Kriger’s place. Accordingly he started early in the forenoon on the
following day.
George and I proceeded to finish the packing and make final
arrangements—a much longer task than we anticipated. There were
so many things that must be done, which we found only at the last
minute, that at 3 p.m., as there was no prospect of getting away until
an hour or so later, I sent George on with the six loaded donkeys,
about thirty of El Hakim’s cattle, and a dozen men, telling him that I
would follow. George rode a mule (of which we had two), which El
Hakim had bought in Abyssinia two years before. They were
splendid animals, and, beyond an inconvenient habit, of which we
never cured them, of shying occasionally and then bolting, they had
no bad points. They generally managed to pick up a living and get fat
in a country where a horse would starve, and, taking them
altogether, they answered admirably in every way. I would not have
exchanged them for half a dozen of the best horses in the
Protectorate. One mule was larger than the other, and lighter in
colour, and was consequently known as n’yumbu m’kubwa, i.e. “the
big mule.” It was used by George and myself as occasion required.
The other, a smaller, darker animal, was known as n’yumbu m’dogo,
i.e. “the little mule.” It was ridden exclusively by El Hakim.
After George’s departure I hurried the remaining men as much as
possible, but it was already dusk when I finally started on my seven-
mile tramp. Some of the men had to be hunted out of the bazaar,
where they had lingered, with their loved ones, in a last long farewell.
There is no twilight in those latitudes (within two degrees of the
equator), so that very soon after our start we were tramping along in
the black darkness. I had no knowledge of the road; only a rough
idea of the general direction. I steered by the aid of a pocket-
compass and a box of matches. After the first hour I noticed that the
men commenced to stagger and lag behind with their lately
unaccustomed burdens, and I had to be continually on the alert to
prevent desertions. I numbered them at intervals, to make sure that
none of them had given me the slip, but an hour and a half after
starting I missed three men with their loads, in spite of all my
precautions. I shouted back into the darkness, and the men
accompanying me did the same, and, after a slight interval we were
relieved to hear an answering shout from the missing men. After
waiting a few moments, we shouted again, and were amazed to find
that the answering shout was much fainter than before. We
continued shouting, but the answers grew gradually fainter and more
faint till they died away altogether. I could not understand it at first,
but the solution gradually dawned upon me. We were on a large
plain, and a few hundred yards to the left of us was a huge belt of
forest, which echoed our shouts to such an extent that the men who
were looking for us were deceived as to our real position, and in their
search were following a path at right angles to our own. I could not
light a fire to guide them, as the grass was very long and dry, and I
should probably have started a bush fire, the consequences of which
would have been terrible. I therefore fired a gun, and was answered
by another shot, seemingly far away over the plain to the right.
Telling the men to sit down and rest themselves on the path, I
ordered Jumbi to follow me, and, after carefully taking my bearings
by compass, started to walk quickly across the plain to intercept
them.
It was by no means a pleasant experience, trotting across those
plains in the pitchy blackness, with the grass up to my waist, and
huge boulders scattered about ready and willing to trip me up. I got
very heated and quite unreasonably angry, and expressed my
feelings to Jumbi very freely. I was in the midst of a violent diatribe
against all natives generally, and Swahili porters in particular, which I
must admit he bore with commendable patience, when the earth
gave way beneath me, and I was precipitated down some apparently
frightful abyss, landing in a heap at the bottom, with all the breath
knocked out of my body. I laid there for a little while, and
endeavoured to collect my scattered faculties. Soon I stood up, and
struck a match, and discovered that I had fallen into an old game-pit,
about 8 feet deep. It was shaped like a cone, with a small opening at
the top, similar to the old-fashioned oubliette. I looked at the floor,
and shuddered when I realized what a narrow squeak I might have
had; for on the centre of the floor were the mouldering remains of a
pointed stake, which had been originally fixed upright in the earth
floor on the place where I had fallen.
“Is Bwana (master) hurt?” said the voice of Jumbi from somewhere
in the black darkness above.
I replied that I was not hurt, but that I could not get out without
assistance; whereupon Jumbi lowered his rifle, and, to the
accompaniment of a vast amount of scrambling and kicking, hauled
me bodily out.
We were by this time very near to the men for whom we were
searching, as we could hear their voices raised in argument about
the path. We stopped and called to them, and presently they joined
us, and we all set off together to join my main party. We reached it
without further mishap, and resumed our interrupted march.
It was very dark indeed. I could not see my hand when I held it a
couple of feet from my face. One of the men happening to remark
that he had been over the path some years before, I immediately
placed him in the van as guide, threatening him with all sorts of pains
and penalties if he did not land us at our destination some time
before midnight.
I was particularly anxious to rejoin George, as I had the tents,
blankets, and food, and he would have a very uninteresting time
without me. We marched, therefore, with renewed vigour, as our
impromptu guide stated that he thought one more hour’s march
would do the business. It didn’t, though. For two solid hours we
groped blindly through belts of forest, across open spaces, and up
and down wooded ravines, until somewhere about eleven p.m.,
when we reached a very large and terribly steep ravine, thickly
clothed with trees, creepers, and dense undergrowth. We could hear
the rushing noise of a considerable volume of water at the bottom,
and in the darkness it sounded very, very far down.
I halted at the top to consider whether to go on or not, but the
thought of George waiting patiently for my appearance with supper
and blankets made me so uncomfortable that I decided to push on if
it took me all night. We thereupon commenced the difficult descent,
but halfway down my doubts as to the advisability of the proceeding
were completely set at rest by one of the men falling down in some
kind of a fit from over-fatigue. The others were little better, so I
reluctantly decided to wait for daylight before proceeding further. I
tried to find something to eat among the multifarious loads, and
fortunately discovered a piece of dry bread that had been thrown in
with the cooking utensils at the last moment. I greedily devoured it,
and, wrapping myself in my blankets, endeavoured to sleep as well
as I was able on a slope of forty-five degrees. A thought concerning
George struck me just before I dropped off to sleep, which comforted
me greatly. “George knows enough to go in when it rains,” I thought.
“He will leave the men with the cattle, and go over to Kriger’s place
and have a hot supper and a soft bed, and all kinds of good things
like that,” and I drew my blankets more closely round me and
shivered, and felt quite annoyed with him when I thought of it.
At daylight we were up and off again, and, descending the ravine,
crossed the river at the bottom, and continued the march. On the
way I shot a guinea-fowl, called by the Swahilis “kanga,” and after an
hour and a half of quick walking I came up with George.
He had passed a miserable night, without food, blankets, or fire,
and, to make matters worse, it had drizzled all night, while he sat on
a stone and kept watch and ward over the cattle. The men who had
accompanied him were so tired that they had refused to build a
boma to keep the cattle in. He seemed very glad to see me. We at
once got the tent put up, a fire made, and the boma built, and soon
made things much more comfortable. In fact, we got quite gay and
festive on the bread and marmalade, washed down with tea, which
formed our breakfast.
El Hakim was at Kriger’s place, about a mile distant. We had to
wait two or three days till he was ready to start, as he had a lot of
private business to transact. We left all the cattle except nine behind,
under Kriger’s charge; we sent the nine back subsequently, as we
found they were more trouble than they were worth.
In the evening I went out to shoot guinea-fowl; at least, I intended
to shoot guinea-fowl, but unfortunately I saw none. I lost myself in
the darkness, and could not find my way back to camp. After
wandering about for some time, I at last spied the flare of the camp
fires, halfway up a slope a mile away, opposite to that on which I
stood. I made towards them, entirely forgetting the small river that
flowed at the foot of the slope. It was most unpleasantly recalled to
my memory as I suddenly stepped off the bank and plunged, with a
splash, waist deep into the icy water. Ugh!
I scrambled up the opposite bank, and reached the camp safely,
though feeling very sorry for myself. El Hakim and George thought it
a good joke. I thought they had a very low sense of humour.
On the following morning George and I sallied forth on sport intent.
George carried the shot-gun, and I the ·303. We saw no birds; but
after an arduous stalk, creeping on all fours through long, wet grass,
I secured a congoni. Congoni is the local name for the hartebeeste
(Bubalis Cokei). The meat was excellent, and much appreciated. El
Hakim joined us in the afternoon, accompanied by Mr. Kriger and Mr.
and Mrs. Knapp, who wished to inspect our camp. We did the
honours with the greatest zest, knowing it would be the last time for
many months that we should see any of our own race.

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Co-Immunoprecipitation Methods for

Brain Tissue Yuji Odagaki

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For further volumes:

ISSN 0893-2336 ISSN 1940-6045 (electronic)

Library of Congress Control Number: 2018962873

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Saskatoon, SK, Canada Wolfgang Walz

The human genome consists of 20,000–30,000 genes coding for 100,000–500,000

Saitama, Japan Yuji Odagaki

Preface to the Series . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v

1 Co-immunoprecipitation Methods for Detection of G Protein-Coupled

10 Isolation and Detection of G Protein-Coupled Receptor (GPCR)

Carolina Aguado • Facultad de Medicina, Departamento de Ciencias Médicas, Instituto

Hiroki Kaneko • Laboratory of Nuclear Transport Dynamics, National Institutes of

Co-immunoprecipitation Methods for Detection

Key words Co-immunoprecipitation, Immunoprecipitation, Pull down, Brain tissue, GPCR

It is known that a significant number of G protein-coupled

as adenosine triphosphate (ATP), via their specific P1 and P2

homogeneous, making it easier to focus on the target genes.

2.1 Animal Eight-week-old Wistar rats were anesthetized with pentobarbital

2.2 Immuno- The tissues were homogenized with a Polytron® homogenizer in

30 min at 4 °C. The pellets were solubilized in ice-cold lysis buffer

3. To avoiding catalytic dissociation by protease during tissue

All steps of the experiments should be performed on ice or in

co-­immunoprecipitation experiments, denaturation of molecules

We successfully demonstrated A1R and P2Y2R hetero-­

Co-immunoprecipitation Methods to Identify Associated

Key words Co-immunoprecipitation, Centrifuge separation, Brain, Estrogen receptor, Postsynaptic

1.1 Co-immuno- Information of protein-protein interactions is very important to

using polymerase chain reaction. This method has also given us a

1.2 Limitation On the other hand, co-immunoprecipitation has some limitations.

1.3 Improving An approach to overcome these problems is using antibodies which

antibody bindings to specific proteins to non-specific proteins in

successfully purified ERα localized at the PSD and identified its

2 Materials and Methods

2.1 Evaluation Before co-immunoprecipitation, our group assessed specificity

can be further separated to the myelin, synaptosomal, and mito-

Trioton-soluble P2: sup sup: S4 (Cytoplasm)

Trioton-insoluble P2: ppt sup: LDM

HDM + LDM (2%)

­ ellet and the supernatant were collected separately as the low-

ERα. It is important that, the triton-insoluble P2 fraction contained

2.3 Co-immuno- Although solubilization of samples is necessary before co-­

triton-insoluble P2 sample by rotating sample tubes overnight at

2.4 Mass For mass spectrometric identification of ERα-associated proteins,

Engaging porters—Characteristics of Swahili, Wa’Nyamwezi, and

Uzi Wa Madini (Wire).

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Saskatoon, SK, Canada Wolfgang Walz

Saitama, Japan Yuji Odagaki

co-immunoprecipitation experiments, denaturation of molecules

We successfully demonstrated A1R and P2Y2R hetero-

ellet and the supernatant were collected separately as the low-

2.3 Co-immuno- Although solubilization of samples is necessary before co-