Table Of ContentNeuromethods 144
Yuji Odagaki
Dasiel O. Borroto-Escuela Editors
Co-Immuno-
precipitation
Methods
for Brain Tissue
N
euromethods
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
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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-p rotein 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,
University of Urbino, Urbino, Italy
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,
University of Urbino, Urbino, Italy
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—
DANDRITE, Aarhus University, Aarhus, Denmark; Department of Biomedicine,
Aarhus University, Aarhus, Denmark
yuji KamiKubo • Department of Pharmacology, Juntendo University School of Medicine,
Tokyo, Japan
xi
