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Methods in
Molecular Biology 2466
B. Vijayalakshmi Ayyar
Sushrut Arora Editors
Affinity
Chromatography
Methods and Protocols
METHODS IN MOLECULAR BIOLOGY
Series Editor
John M. Walker
School of Life and Medical Sciences
University of Hertfordshire
Hatfield, Hertfordshire, UK
For further volumes:

http://www.springer.com/series/7651
For over 35 years, biological scientists have come to rely on the research protocols and
methodologies in the critically acclaimed Methods in Molecular Biology series. The series was
the first to introduce the step-by-step protocols approach that has become the standard in all
biomedical protocol publishing. Each protocol is provided in readily-reproducible step-by-
step fashion, opening with an introductory overview, a list of the materials and reagents
needed to complete the experiment, and followed by a detailed procedure that is supported
with a helpful notes section offering tips and tricks of the trade as well as troubleshooting
advice. These hallmark features were introduced by series editor Dr. John Walker and
constitute the key ingredient in each and every volume of the Methods in Molecular Biology
series. Tested and trusted, comprehensive and reliable, all protocols from the series are
indexed in PubMed.
Affinity Chromatography
Methods and Protocols
Edited by
B. Vijayalakshmi Ayyar
Molecular Virology and Microbiology, Baylor College of Medicine, Houston, TX, USA
Sushrut Arora
Astero Erado Inc, Houston, TX, USA
Editors
B. Vijayalakshmi Ayyar Sushrut Arora
Molecular Virology and Microbiology Astero Erado Inc
Baylor College of Medicine Houston, TX, USA
Houston, TX, USA
ISSN 1064-3745 ISSN 1940-6029 (electronic)

Methods in Molecular Biology
ISBN 978-1-0716-2175-2 ISBN 978-1-0716-2176-9 (eBook)
https://doi.org/10.1007/978-1-0716-2176-9
© Springer Science+Business Media, LLC, part of Springer Nature 2022

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,
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Preface
Affinity chromatography is a versatile biochemical separation technique which relies on

reversible interaction between a target molecule and its cognate ligand. Traditional purifica-
tion systems typically involve multiple steps which require long processing times leading to
increased costs and limited flexibility. Affinity chromatography addresses many of these
constraints due to its selectivity and specificity toward the target accompanied with the
ease of operation and optimization. This technique has come a long way since its discovery
five decades ago due to groundbreaking advancements in both methodology and technol-
ogy which led to its widespread application in many fields from lab bench to large-scale
manufacturing to miniaturized microfluidic devices. Affinity chromatography is extensively
used not only for purification of biomolecules and biopharmaceuticals but also as an
analytical tool to isolate, identify, and measure targets and/or characterize specific interac-
tions in various basic and applied studies.
The success of any technique relies on the depth of knowledge and understanding of the
procedure which empowers the user with the confidence to use appropriate resources and
optimizations to achieve satisfactory result. In terms of affinity chromatography, it is
imperative to have a basic understanding of the nature of interactions between the target
and the ligand to select appropriate affinity ligand and purification procedure in order to
achieve maximum recovery of the target. Keeping this in mind, this book was designed to
provide the reader with methodologies to purify a diverse array of molecular targets such as
antibodies, extracellular vesicles, recombinant proteins, biomarkers, metabolites, plant
organelles, and nucleic acids. The book will also discuss ligand identification and selection
along with protocols on building affinity matrix using entrapment, covalent immobilization,
and molecular imprinting techniques.
We would like to express our gratitude to the senior editor, Prof. John Walker, who
guided us patiently throughout the process. We are grateful to all the contributors for
sharing their expertise and waiting patiently for all the contents to get assimilated and
published. We are sure that the protocols containing the step-by-step procedure along
with experience-based tips and tricks and troubleshooting advice will prove to be an
invaluable resource to anyone employing affinity chromatography-based methodologies.
Houston, TX, USA B. Vijayalakshmi Ayyar

Sushrut Arora
v
Contents
Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v
Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix
PART I APPLICATIONS OF AFFINITY CHROMATOGRAPHY

1 Antibody Purification Using Affinity Chromatography . . . . . . . . . . . . . . . . . . . . . . 3
Arabelle Cassedy and Richard O’Kennedy
2 TIM4-Affinity Methods Targeting Phosphatidylserine
for Isolation or Detection of Extracellular Vesicles . . . . . . . . . . . . . . . . . . . . . . . . . . 23
Takeshi Yoshida and Rikinari Hanayama
3 Application of Affitins for Affinity Purification of Proteins. . . . . . . . . . . . . . . . . . . . 37
Barbara Mouratou and Frédéric Pecorari
4 Identification of Clusterin as a Major ABri- and ADan-Binding
Protein Using Affinity Chromatography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49
Agueda Rostagno, Miguel Calero, and Jorge Ghiso
5 Application of a Novel CL7/Im7 Affinity System in Purification
of Complex and Pharmaceutical Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61
Louise T. Chow and Dmitry G. Vassylyev
6 Positive and Negative Affinity Chromatography to Purify Norovirus
P-Domain Protein . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83
Wilhelm Salmen
7 Robotic Affinity Purification of Soluble and Insoluble Recombinant
Glutathione-S-Transferase Fusion Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93
Robert O. J. Weinzierl
8 Application of Immunoaffinity Mass Spectrometry (IA-MS)
for Protein Biomarker Quantification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111
Joe Palandra, Nikolaos Psychogios, and Hendrik Neubert
9 Rapid Affinity Purification of Tagged Plant Mitochondria (Mito-AP). . . . . . . . . . 121
Markus Niehaus and Marco Herde
10 Arginine-Affinity Chromatography for Nucleic Acid (DNA
and RNA) Isolation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 135
Rita Carapito, Joana F. A. Valente, João A. Queiroz,
and Fani Sousa
11 Purification and Analysis of Nucleotides and Nucleosides
from Plants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 145
Henryk Straube and Marco Herde
vii
viii Contents
PART II LIGAND SELECTION AND AFFINITY MATRIX

PREPARATION FOR AFFINITY CHROMATOGRAPHY
12 Ligand Selection for Affinity Chromatography Using Phage Display . . . . . . . . . . 159

Krištof Bozovičar, Peter Molek, Barbara Jenko Bizjan,
and Tomaž Bratkovič
13 Selection and Application of Aptamer Affinity for Protein Purification . . . . . . . . . 187
Ana Paula de Jesus Santos, Ágatha Oliveira-Giacomelli,
Vanessa Karen de Sá, Isis Cristina do Nascimento,
Erika de Simone Molina, and Henning Ulrich
14 Entrapment of Proteins Within Columns for High-Performance
Affinity Chromatography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 205
Saumen Poddar, Sadia Sharmeen, and David S. Hage
15 Preparation of Affinity Chromatography Monolith in Miniaturized
Format and Application for Protein Separation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 229
Kishore K. R. Tetala
16 Ligand Immobilization Methods for Affinity Chromatography . . . . . . . . . . . . . . . 241
Chandra K. Dixit
17 Preparation of Molecularly Imprinted Poly(N-Isopropylacrylamide)
Thermosensitive Based Cryogels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 249
Işık Perçin, Neslihan Idil, and Adil Denizli
18 Preparation of Staphylococcal Protein A Imprinted Supermacroporous
Cryogel Beads . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 261
Sevgi Aslıyüce, Bo Mattiasson, and Adil Denizli
Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 275
Contributors
SEVGI ASLIYÜCE • Department of Chemistry, Hacettepe University, Ankara, Turkey

BARBARA JENKO BIZJAN • Clinical Institute of Special Laboratory Diagnostics, University
Children’s Hospital, University Medical Center, Ljubljana, Slovenia
KRIŠTOF BOZOVIČAR • Department of Pharmaceutical Biology, Faculty of Pharmacy,
University of Ljubljana, Ljubljana, Slovenia
TOMAŽ BRATKOVIČ • Department of Pharmaceutical Biology, Faculty of Pharmacy,
University of Ljubljana, Ljubljana, Slovenia
MIGUEL CALERO • Instituto de Salud Carlos III, Madrid, Spain; Network Center for
Biomedical Research in Neurodegenerative Diseases (CIBERNED), Madrid, Spain;
Alzheimer’s Center Reina Sofia Foundation – CIEN Foundation, Madrid, Spain
RITA CARAPITO • CICS-UBI – Health Sciences Research Centre, University of Beira Interior,
Covilhã, Portugal
ARABELLE CASSEDY • School of Biotechnology, Dublin City University, Dublin, Ireland
LOUISE T. CHOW • Department of Biochemistry and Molecular Genetics, Schools of Medicine
and Dentistry, University of Alabama at Birmingham, Birmingham, AL, USA
ANA PAULA DE JESUS SANTOS • Departamento de Bioquı́mica, Instituto de Quı́mica,
Universidade de São Paulo, São Paulo, Brazil
VANESSA KAREN DE SÁ • Departamento de Bioquı́mica, Instituto de Quı́mica, Universidade
de São Paulo, São Paulo, Brazil
ERIKA DE SIMONE MOLINA • Departamento de Bioquı́mica, Instituto de Quı́mica,
ADIL DENIZLI • Department of Chemistry, Hacettepe University, Ankara, Turkey
CHANDRA K. DIXIT • Lumos Diagnostics, Sarasota, FL, USA
ISIS CRISTINA DO NASCIMENTO • Departamento de Bioquı́mica, Instituto de Quı́mica,
JORGE GHISO • Department of Pathology, New York University School of Medicine, New York,
NY, USA; Department of Psychiatry, New York University School of Medicine, New York,
NY, USA
DAVID S. HAGE • Department of Chemistry, University of Nebraska-Lincoln, Lincoln, NE,
USA
RIKINARI HANAYAMA • Department of Immunology, Kanazawa University Graduate School
of Medical Sciences, Kanazawa, Ishikawa, Japan; WPI Nano Life Science Institute
(NanoLSI), Kanazawa University, Kanazawa, Ishikawa, Japan
MARCO HERDE • Department of Molecular Nutrition and Biochemistry of Plants, Leibniz
Universit€at Hannover, Hannover, Germany
NESLIHAN IDIL • Department of Biology, Hacettepe University, Ankara, Turkey
BO MATTIASSON • Department of Biotechnology, Lund University, Lund, Sweden
PETER MOLEK • Department of Pharmaceutical Biology, Faculty of Pharmacy, University of
Ljubljana, Ljubljana, Slovenia
BARBARA MOURATOU • Nantes Université, Univ Angers, INSERM, CNRS, Immunology
and New Concepts in ImmunoTherapy, INCIT, UMR 1302/EMR6001, Nantes, France
HENDRIK NEUBERT • Pfizer Worldwide Research Development and Medical, Andover, MA,
USA
ix
x Contributors
MARKUS NIEHAUS • Department of Molecular Nutrition and Biochemistry of Plants, Leibniz

RICHARD O’KENNEDY • School of Biotechnology, Dublin City University, Dublin, Ireland;
Hamad Bin Khalifa University, Doha, Qatar; Qatar Foundation, Doha, Qatar
ÁGATHA OLIVEIRA-GIACOMELLI • Departamento de Bioquı́mica, Instituto de Quı́mica,
JOE PALANDRA • Pfizer Worldwide Research Development and Medical, Andover, MA, USA
FRÉDÉRIC PECORARI • Nantes Université, Univ Angers, INSERM, CNRS, Immunology and
New Concepts in ImmunoTherapy, INCIT, UMR 1302/EMR6001, Nantes, France
IŞIK PERÇIN • Department of Biology, Hacettepe University, Ankara, Turkey
SAUMEN PODDAR • Department of Chemistry, University of Nebraska-Lincoln, Lincoln, NE,
USA
NIKOLAOS PSYCHOGIOS • Pfizer Worldwide Research Development and Medical, Andover,
MA, USA
JOÃO A. QUEIROZ • CICS-UBI – Health Sciences Research Centre, University of Beira
Interior, Covilhã, Portugal
AGUEDA ROSTAGNO • Department of Pathology, New York University School of Medicine, New
York, NY, USA
WILHELM SALMEN • Department of Molecular Virology and Microbiology, Baylor College of
Medicine, Houston, TX, USA
SADIA SHARMEEN • Department of Chemistry, University of Nebraska-Lincoln, Lincoln, NE,
USA
FANI SOUSA • CICS-UBI – Health Sciences Research Centre, University of Beira Interior,
Covilhã, Portugal
HENRYK STRAUBE • Department of Molecular Nutrition and Biochemistry of Plants, Leibniz
KISHORE K. R. TETALA • Centre for Bioseparation Technology (CBST), Vellore Institute of
Technology (VIT), Vellore, Tamil Nadu, India
HENNING ULRICH • Departamento de Bioquı́mica, Instituto de Quı́mica, Universidade de
São Paulo, São Paulo, Brazil
JOANA F. A. VALENTE • CDRSP-IPLEIRIA - Centre for Rapid and Sustainable Product
Development, Instituto Politécnico de Leiria, Marinha Grande, Portugal
DMITRY G. VASSYLYEV • Department of Biochemistry and Molecular Genetics, Schools of
Medicine and Dentistry, University of Alabama at Birmingham, Birmingham, AL, USA
ROBERT O. J. WEINZIERL • Department of Life Sciences, Imperial College London, London,
UK
TAKESHI YOSHIDA • Department of Immunology, Kanazawa University Graduate School of
Medical Sciences, Kanazawa, Ishikawa, Japan; WPI Nano Life Science Institute
(NanoLSI), Kanazawa University, Kanazawa, Ishikawa, Japan
Part I
Applications of Affinity Chromatography

Chapter 1
Antibody Purification Using Affinity Chromatography

Arabelle Cassedy and Richard O’Kennedy
Abstract
Antibodies are an integral part of many biological assays and biotherapeutics. However, the sources from
which antibodies are derived frequently contain other contaminants which may interfere with assays or
cause adverse reactions if administered in vivo. Therefore, a means of isolating these antibodies from their
source at high levels of purity is critical. Affinity chromatography is currently one of the most widely applied
methods for the purification of antibodies. This method relies on specific and reversible, interactions
between antibody structures, or recombinant tags fused to these structures, and ligands immobilized on
solid support matrices, generally within a column. Herein, common chromatographic methods applied to
antibody purification are described. These include the purification of IgG, and its recombinant forms,
through protein A, protein G and immobilized metal affinity chromatography.
Key words Affinity purification, Antibodies, Chromatography, Methods, Recombinant proteins
1 Introduction
Antibodies are members of the immunoglobulin family. These

naturally occurring glycoproteins play an essential role in the
immune system, predominantly in the detection of invading patho-
gens. However, the detection capabilities of antibodies have since
been modified and tailored for implementation in numerous
research, clinical and other applications. They are now used exten-
sively, both in vivo and in vitro, for a host of applications including
diagnostics, therapeutics, analytical techniques, immunoaffinity
purifications, food- and water-borne pathogen detection, and vari-
ous other forms of immunosensing [1–4]. The demand for com-
mercially available, approved antibodies is estimated to increase,
with a higher number of antibodies predicted to be in late-stage
evaluation studies, especially for potential clinical utilization, in
2020 than any prior year [5]. Given the current and increasing
popularity of antibodies, it is necessary to have efficient methods
for their purification from the range of sources wherein they are
produced.
B. Vijayalakshmi Ayyar and Sushrut Arora (eds.), Affinity Chromatography: Methods and Protocols, Methods in Molecular Biology,
vol. 2466, https://doi.org/10.1007/978-1-0716-2176-9_1, © Springer Science+Business Media, LLC, part of Springer Nature 2022
3
4 Arabelle Cassedy and Richard O’Kennedy
Previously, antibodies were largely isolated from the body fluids

and tissues of immunized animals; however, with the advent of
recombinant protein expression and phage display, these proteins
may now also be found in a much wider range of matrices. Exam-
ples include ascites fluid (no longer used commonplace), serum,
plasma, egg yolk, mammalian and hybridoma cell culture media,
bacterial cell cultures, cell suspensions, cell-free systems, and plant
tissue [6, 7]. Naturally, many of these sources are complex and
contain contaminant proteins which must be removed in order to
yield a homogeneous and pure antibody preparation, a requirement
for the majority of antibody applications. There are a multitude of
chromatographical methods by which antibodies can be isolated
from their respective source. These are largely based on the bio-
chemical properties of the protein, for example, size, shape, charge,
isoelectric point, or hydrophobicity. However, one of the most
effective and commonly employed methods is affinity
chromatography [8].
Affinity chromatography is a selective, reversible, liquid chro-
matography–based purification method. The first report of such a
chromatographic method was by Emil Starkenstein in 1910,
whereby the α-amylase enzyme was isolated using a solid form of
its substrate, starch [9]. In terms of antibody purification, it was
Campbell, Luescher and Lerman (1951) who were the first to
report an immunoaffinity separation. This research detailed the
purification of anti-bovine serum albumin (BSA) antibodies from
rabbit serum via the use of a BSA antigen immobilized onto an
activated cellulose matrix [10]. Efforts to effectively purify proteins
via ligands immobilized on various matrices continued until 1968,
when Cuatrecasas, Wilchek, and Anfinsen (1968) made a critical
advancement in the affinity chromatography field. They employed a
novel beaded agarose, Sepharose, alongside a recently developed
cyanogen bromide–mediated covalent protein coupling method to
generate a stable Sepharose matrix, coupled with various enzyme
substrates and inhibitors [11–13]. Through this, they demon-
strated the effective chromatographic purification of enzymes in a
column/resin format. Due to the efficiency and adaptability of this
method, its uptake was widespread, leading the way in what has
now become known as affinity chromatography.
Classically, affinity chromatography involves the biological pro-
cess of a macromolecule binding to its cognate ligand, for example,
the binding of an antibody to its cognate antigen, a lectin to its
sugar or an enzyme to its substrate. However, the term has since
broadened to encompass alternative binding pairs, and one of the
most prominent examples of this is the binding of histidine-tagged
proteins to immobilized metal ions in immobilized metal affinity
chromatography (IMAC) [14]. The underlying principle of affinity
chromatography is the initial immobilization of the ligand, or other
binding entity, onto the stationary phase. The antibody-containing
Antibody Purification Using Affinity Chromatography 5
Precipitation
Pre-Purification Affinity Post-Purification

Treatment Purification Treatment
Volume Reduction
Antibody Sample
Buffer Exchange
Application Binding Washing Elution
Pelleting/Lysing Cells
Fig. 1 Sample Workflow for an Affinity Purification. Prior to purification, the antibody-containing sample
generally requires some level of pretreatment. Examples of pretreatments include clarification of the sample,
precipitation of proteins or reduction of the sample volume. Once prepared, the sample is then applied to a
column where the antibody is captured on a specific resin, for example, Protein A resin. A series of wash steps
can be used to remove contaminant proteins, after which, the purified antibody sample is eluted. Further
treatment, such as exchanging into an alternative buffer, may be required post-purification depending on the
intended future application of the purified antibody
solution is then applied via a mobile phase which passes over the
stationary phase, and this is where binding of the antibody occurs.
Here, the antibody interacts with the ligand and is retained, while
the remainder of the contaminants are removed through a series of
wash steps. Antibody which has remained bound to the stationary
phase may then be eluted. This general workflow for an immunoaf-
finity purification is depicted in Fig. 1. The elution method varies
depending on the style of affinity chromatography being imple-
mented. The purification method of choice is typically dictated by
the type and format of antibody being purified.
1.1 Antibody Antibodies may be generally categorized into three groups, poly-
Formats clonal, monoclonal, and recombinant. Polyclonal refers to a mix-
ture of antibodies against a given target. These antibodies are
produced by multiple B-cells, and as such, will have varying affi-
nities to a range of epitopes on the target molecule. Conversely,
monoclonal antibodies are produced by one B-cell and are typified
by singular epitope targeting. Recombinant antibodies are prepared
using – in vitro methods. Briefly, the antibody genes are isolated
through polymerase chain reaction (PCR). Thereafter, the genes
are cloned into vectors and transformed into a host organism, for
example, E. coli, or yeast, to generate an antibody library. Target-
specific antibodies are identified from this library using various
methods, for example phage display [15]. Classically, recombinant
antibodies were generated to detect one epitope on the target, in a
Fa
b
F(ab’)2
F(ab)
Fc
IgG sdAb
scAb
scFv
Diabody
Minibody
ScFv-Fc
Chain Legend
Variable Light Constant Light Variable Heavy Constant Heavy
Fig. 2 Examples of Various Antibody Formats. The “whole” antibody molecule, IgG, consists of two heavy and
two light chains. The heavy chain may be further subdivided into three constant regions and one variable
region, while the light chain is separated into one constant and one variable region. Recombinant antibodies
are fragments of this “whole” molecule and comprise a combination of variable and constant regions from the
heavy and light chains. The incorporation of constant regions into recombinant fragments generally imparts a
greater stability to the molecule. Examples of recombinant formats containing constant regions include the F
(ab0 )2, F(ab), scAb, and minibody. The addition of certain constant regions (Fc) to the constructs also offers Fc
functionality, which is useful for purifications or other in-vivo applications, an example of this can be seen in
the scFv-Fc format. The chains within recombinant forms are typically joined by either disulfide bonds or linker
molecules, such as the commonly used glycine–serine linker
similar fashion to the monoclonal antibody. However, develop-

ments in protein engineering technology have since facilitated the
generation of multispecific recombinant antibodies which bind
multiple different epitopes simultaneously [16]. Recombinant anti-
bodies are available in a wide range of formats, each offering
varying attributes, for example solubility, stability, and varying
half-lives (Fig. 2) [17].
The format of the antibody also plays large a role in determin-
ing which purification method should be employed. For instance,
Protein A/G chromatography relies predominantly on the binding
of these bacterial proteins to the Fc portion of the antibody, with
the exception of protein A binding the variable heavy chain belong-
ing to the human VH3 family [18, 19]. However, some recombi-
nant antibody fragments do not contain Fc regions, therefore, this
widely used method is inappropriate for many of the recombinant

antibody classes. Similarly, antibodies produced in hybridoma cul-
ture typically do not contain additional tags on the protein, there-
fore, methods such as IMAC are not applicable. While antibody
format plays a role in dictating which purification strategy is used,
other important deciding factors are antibody isotype, subclass and
species. Each of these factors can greatly affect purification effi-
ciency; therefore, it is imperative to characterize the antibody sam-
ple prior to purification.
1.2 Antibody When purifying monoclonal antibodies, knowledge of the subclass

Characterization (isotype) and species of the immunoglobulin is essential for deter-
mining which affinity ligand to use. There are several methods
which can be employed in order to identify isotype; however, two
of the most popular methods are lateral flow immunoassay (LFIA)
and enzyme-linked immunosorbent assay (ELISA). There are also
commercial kits available to identify light chain variants, that is, κ or
λ. Titration of the antibody sample against the antigen of interest is
also commonly used in initial characterization steps. This reflects
the level of antibody activity in the source. This titer can be further
used as a reference for postpurification to determine the success of
the method. Once the sample is characterized to an acceptable
level, contaminants within the samples source must be then be
removed prior to purification in a process termed clarification.
1.3 Sample As aforementioned, antibodies are found in a range of sources,

Clarification most of which contain cellular and molecular contaminants.
These contaminants are introduced either in the form of growth
media components or as by-products of host cells. The contami-
nants must be removed or reduced prior to purification as their
presence can clog purification columns or compete for affinity
ligand binding, rendering the overall purification less efficient.
Removal of contaminants is also highly important for the purifica-
tion of antibodies destined for in vivo applications. The presence of
such components could cause undesirable consequences, such as
adverse immune reactions in the patient [20]. Commonplace clar-
ification methods include centrifugation and filtration of the cell
lysates. Centrifugation of the sample should be performed first.
Without a centrifugation step, excess solid matter may clog filter
pores and cause loss of valuable sample. These two methods pri-
marily act to remove cellular debris, while alternative methods, such
as buffer exchange, act to remove media components [21]. Post–
sample clarification, the volume of the sample may need to be
reduced to provide a more manageable quantity for purification.
For example, in hybridoma cultures where the volume of media
(containing the antibody) would be too high to permit benchtop
purification without any preconcentration step. Volume reduction
is also useful when there is a need to concentrate the amount of
antibody in a given sample.
1.4 Reduction of Precipitation involves the addition of salt to the sample to the point
Sample Volume where the ionic strength of the solution renders proteins insoluble.
This leads to protein–protein interactions, resulting in aggregation
1.4.1 Precipitation
and subsequent precipitation of the target, known as “salting-out.”
One of the most commonly used salts is ammonium sulfate. The
salt concentration at which a protein will precipitate at varies
depending on the proteins’ isoelectric point, making this a useful
method for isolating particular targets. Precipitation can be positive
or negative depending upon what is precipitated. For example, in
terms of antibodies, precipitation can be used to isolate only anti-
bodies, termed positive precipitation, or to precipitate out other
undesired proteins, which is known as negative precipitation
[22]. A drawback of this method is that usually other undesired
proteins will “salt-out” at similar ionic strengths to the target,
meaning that further cleanup steps are typically required. Precipita-
tion is also a useful method for purification and concentration of
antibodies such as avian IgY, which does not interact with the
typical affinity purification proteins A or G [23, 24].
1.4.2 Sample Another alternative for sample clarification and reduction of sample
Concentration volume is to employ concentration systems using filter membranes.
The filter permits proteins smaller than the pore size to pass
through, along with excess media, while proteins with molecular
weights above the cutoff are retained in the remaining concentrated
media [25]. Once the protein has been concentrated to the desired
volume, the process may proceed to the next step, affinity
purification.
1.5 Affinity Proteins A and G are a class of bacterial protein which bind to the Fc
Chromatography region of a wide range of immunoglobulin species and isotypes.
Protein A, derived from Staphylococcus aureus, also binds the
1.5.1 Proteins A, G, A/G,
human heavy chain of the VH3 family. Protein A is the most widely
and L
used affinity ligand as it shows high stability and high specificity for
IgG [26]. While protein A is routinely used for most antibody
purifications, it does not exhibit binding to all immunoglobulin
species and classes, for example, human IgG3 and rat IgG, where
protein G must be used. Protein G was isolated from Streptococcus
and demonstrates binding to the Fc regions of immunoglobulins;
however, protein G is also known to bind other proteins such as
albumin or α2-macroglobulin. Protein engineering methods have
since led to the abolition of such undesired binding regions in
protein G [27, 28]. The binding capacity of both protein A and
G for IgG (or its equivalent) varies depending on the Ig class and
species. It is good practice to refer to information provided by the
resin manufacturers to ensure the most suitable resin is employed
for the relevant antibody. To overcome the differences in selectivity
for antibody subclasses and species, a chimeric form of proteins A
and G was developed. Termed protein A/G, this fusion protein

offers broad-spectrum binding to most isotypes and species [29].
Proteins A, G, and their fusion protein, are all widely used for
the purification of “whole” antibody molecules. However, the
advent of recombinant antibody fragment generation, and the
discovery of new types of antibody, such as the camelid (VHH)
and shark (IgNar) antibodies, which are devoid of Fc regions,
necessitated new affinity ligands to bind antibody regions other
than the Fc. Protein L, another bacterial protein, sourced from
Peptostreptococcus magnus, was found to bind the kappa variable
light chains, present in roughly two thirds of mammalian immuno-
globulins [30]. This capacity to bind the light chain also makes
protein L favorable in the purification of fragments such as scFv,
scAb, and Fab proteins. Although protein L does offer an alterna-
tive in situations where protein A and G are unsuitable, there
remains the issue of purification of antibodies which do not contain
kappa light chains, or alternatively, lack light chains entirely. To
overcome this, peptide/protein tags are incorporated with the
antibody genes to facilitate purification through other affinity
methods, one of the most popular being immobilized metal affinity
chromatography (IMAC).
1.5.2 Immobilized Metal In contrast to the previously described affinity chromatographic

Affinity Chromatography methods, IMAC employs an immobilized metal ion as the affinity
ligand, in lieu of a protein. Chelating agents, such as iminodiacetic
acid (IDA) or nitrilotriacetic acid (NTA), coordinate the metal ions
to the solid chromatography matrix where the affinity reaction
occurs. The presence of electron donor atoms on the side chains
or termini of amino acids such as histidine, serine, aspartate, cyste-
ine, glutamate, methionine, tyrosine, and tryptophan have affinity
toward the immobilized metal ion, resulting in binding to the
IMAC resin. While many amino acids present with this metal
affinity, histidine is shown to possess the highest retention rate.
Due to this tags consisting of multiple consecutive histidine resi-
dues (typically hexahistidine) are now routinely expressed at the N-
and C-terminals of recombinant proteins to facilitate stronger
interactions of the recombinant protein with the IMAC resin. A
drawback to the use of IMAC is the fact that many E. coli host
proteins adhere to the metals employed in IMAC [31]. Hence,
supplementary polishing steps, such as size exclusion chromatogra-
phy, are often required after IMAC purification of recombinant
proteins expressed in E. coli systems.
There is a range of metal ions employed in IMAC, including
Ni2+, Zn2+, Co2+, Cu2+, and Fe2+. Each has unique properties in
terms of specificity and affinity for histidine. The affinities of pro-
teins for the metal ions are ranked as Cu2+ > Ni2+ > Zn2+ > Co2+.
The choice of metal ion is dictated by the nature of the purification;
for example, Zn2+ may be chosen when purifying bacterial proteins
as it has a lower affinity for the E. coli host proteins [32]. However,
this metal ion also has a lower affinity for the histidine tag, and
purification through this means may result in a lower yield of the
protein of interest. If a high yield of protein is a desired outcome of
the process, it may be more favorable to perform IMAC using Ni2+
resin, which has a higher binding affinity with histidine, and per-
form cleanup steps to remove remaining contaminant host
proteins.
In addition to protein A, protein G, and IMAC resins, there are
multiple other ligands which can be used to purify antibodies. Some
examples of these include the use of antigens immobilized on solid
surfaces to immunoaffinity purify antibody mixtures. Alternatively,
smaller peptides which correlate to specific regions on the antigen
can be used. This method can be particularly useful for polyclonal
antibody preparations where only a portion of the antibodies pres-
ent in the mixture will be specific for the target antigen
[33]. Branched and cyclic peptides are another alternative for
antibody-binding ligands. Some of these peptides are referred to
as mimetic ligands as they present binding similar to already estab-
lished immunoglobulin binding proteins such as protein A
[34]. Further examples of alternative ligands are anti-species/
anti-tag antibodies, affimers, aptamers, designed ankaryn repeat
proteins, monobodies, affitins, thiophilic ligands, and lectins [35–
37]. While there are numerous ligand options for the purification of
antibodies, this chapter will focus primarily on protein A/G/L and
IMAC purification strategies as their application is widespread and
such ligands are suitable for the purification of most antibodies and
antibody constructs.
2 Materials
Chemicals can be sourced from Sigma-Aldrich, unless otherwise

stated. Buffers and reagents are typically prepared using
dH2O. These should be checked for signs of precipitation or con-
tamination before use.
2.1 Antibody 1. There are multiple commercial kits available, for example, the
Isotyping Rapid Antibody Isotyping Kit Plus Kappa and Lambda for
Mouse (Pierce™) or IsoStrip™ Mouse Monoclonal Antibody
2.1.1 Lateral Flow
Isotyping Kit from Roche (see Note 1). These kits are stored
refrigerated between 2 and 8 C. These kits contain lateral flow
strips featuring immobilized anti-isotype antibodies, for exam-
ple, IgG1, IgG2a, IgG2b, IgG3, IgM, and IgA. There are also
test lines which detect the light chain on the monoclonal anti-
bodies, allowing the determination of the light chain class (κ or
λ). A sample diluent is also typically provided in these kits.
2.1.2 ELISA 1. Commercial isotyping ELISA kits, for example, Invitrogen’s Ig

Isotyping Mouse Instant ELISA Kit (see Note 1). This kit can
be stored in its entirety at 20 C. Contained within are anti-
isotype antibodies (e.g. anti- IgG1, IgG2a, IgG2b, IgG3, IgA,
IgM, light chain specific: κ or λ), a HRP-conjugated anti-
mouse pAb, positive controls, sample diluent, wash buffers,
HRP substrate, and stop solution.
2. dH2O.
2.2 Sample 1. Centrifuge.

Clarification
2.2.1 Clarification of
Mammalian Cell Culture
(Hybridoma)
2.2.2 Clarification of 1. Centrifuge.

Serum 2. Membrane filters (0.22 μm pore size).
2.2.3 Clarification of 1. Centrifuge.

Bacterial Cultures 2. Sonication/equilibration buffer (50 mM NaH2PO4, 300 mM
NaCl, 10 mM imidazole, adjust to 1 L with dH2O, pH 8.0).
Protease inhibitors may be added to this directly before
clarification.
3. Cell sonicator.
4. Membrane filters (0.22 μm pore size).
2.3 Reduction of 1. Saturated ammonium sulfate solution (see Note 2).

Sample Volume 2. Phosphate-buffered saline (PBS; 137 mM NaCl, 2.7 mM KCl,
2.3.1 Ammonium Sulfate 12 mM Na2HPO4, 1.2 mM KH2PO4, make to 1 L with dH2O
Precipitation and adjust to pH 7.4).
2.3.2 IgY Purification 1. Centrifuge.

2. Commercial IgY purification kit, for example, the Pierce™
Chicken IgY Purification Kit which is stored at 4 C. This kit
contains the delipidation and precipitations buffers required for
the isolation of IgY from egg yolk.
3. PBS: as described under Subheading 2.3.1.
2.3.3 Sample 1. Concentration system, for example, Amicon® Stirred Ultrafil-

Concentration tration Cell (Millipore).
2. Molecular weight cutoff membranes.
2.4 Purification of 1. Polypropylene disposable columns, 20 mL capacity (Bio-Rad).

Antibodies Using 2. Protein A agarose, protein G agarose, protein A/G agarose,
Affinity protein L agarose (Pierce) (see Note 3).
Chromatography
3. Proteins A, G, A/G and L equilibration/binding buffer: PBS
2.4.1 Proteins A, G, and L (Subheading 2.3.1).
Chromatography 4. Elution buffer: 100 mM Glycine–HCl, pH 2.7.
5. Neutralization buffer: 1 M Tris–HCl, pH 8.5.
6. Resin storage buffer: Includes 0.02% (w/v) sodium azide
prepared in dH2O. Exert caution when using azide as it is
acutely toxic.
2.4.2 Immobilized Metal 1. Polypropylene disposable columns, 20 mL capacity.

Affinity Chromatography 2. IMAC resin, for example, Ni-NTA.
3. Sonication/equilibration buffer (as described under Subhead-
ing 2.2.3; see Notes 4 and 5).
4. Wash Buffer 1: 50 mM NaH2PO4, 300 mM NaCl, 20 mM
imidazole, adjust to 1 L with dH2O, pH 8.0 (see Notes 5 and
6).
5. Wash Buffer 2: 50 mM NaH2PO4, 300 mM NaCl, 30 mM
imidazole, adjust to 1 L with dH2O, pH 8.0 (see Notes 5 and
6).
6. Elution buffer 50 mM NaH2PO4, 300 mM NaCl, 300 mM
imidazole (for hexahistidine), adjust to 1 L with dH2O,
pH 8.0.
2.4.3 Buffer Exchange 1. Centrifuge.

2. Molecular weight cutoff concentrator columns, for example,
Vivaspin range.
3. PBS: as described under Subheading 2.3.1.
3 Methods
This section describes the various steps involved in each part of the
purification process, that is, clarification, volume adjustment, and
purification. These protocols are a general recommendation for
each of these methods; however, some adjustments may be
required depending on the antibody to be purified and the overall
requirements of the purification.
3.1 Antibody As previously mentioned, characterization of the antibodies within

Isotyping the sample to be purified is an essential step. This is with particular
note for monoclonal antibodies, as factors such as isotype and chain
class greatly affect the subsequent choice of affinity ligand.
Isotyping is not as pertinent when purifying recombinant antibo-

dies as these proteins are generally expressed fused to a histidine
tag. This tag facilitates purification via IMAC and functions, irre-
spective of antibody format, isotype, or class. If a scenario arises
where the isotype or class of an antibody is unknown, then fusion
ligands such as A/G are an option, as this fusion binds most
antibodies.
3.1.1 Lateral Flow 1. Prepare sample as per the manufacturer’s guidelines, for exam-
ple, dilute tissue culture supernatant 1:100 with sample
diluent.
2. Equilibrate lateral flow cassettes to room temperature. Each
cassette will have test lines containing immobilized anti-isotype
and anti-light chain class antibodies.
3. Add the sample to be tested to each well of the cassette.
4. After 5–10 min determine the result on the strip. Disregard any
result after 10 min.
5. Results are analyzed by first confirming the presence of a col-
ored line at the control, indicating a successful assay. Secondly,
the isotype and class of the antibody is determined by identify-
ing the darkest colored test line bands on the lateral flow strip.
Each test line will have an associated isotype, thus allowing for
elucidation of isotype.
3.1.2 ELISA 1. Prepare the wash buffer and positive controls in dH2O, as per
manufacturers’ guidelines.
2. Remove plates from 20 C and use immediately.
3. Add 140 μL of dH2O to the lyophilized pellets in the sample
wells and negative control wells Similarly, add 100 μL dH2O to
the positive control wells.
4. Add 50 μL of positive control (lyophilized IgG1, IgG2a,
IgG2b, IgG3, IgA, IgM, κ, and λ isotypes) and 10 μL negative
control (e.g., tissue culture media) or sample (e.g., hybridoma
supernatant) to the appropriate wells.
5. Cover the plate with adhesive film and incubate at room tem-
perature for 2 h, under agitation on a microplate shaker.
6. Remove film, empty wells and wash six times with 400 μL wash
buffer. Avoid touching the bottom of the wells when washing.
7. Add 100 μL of TMB substrate to each well and incubate at
room temperature for 10–15 min, away from direct sunlight.
8. Add 100 μL Stop Solution to all wells.
9. Read absorbances immediately on a plate spectrophotometer at
an absorbance 450 nm. The range 610-650 nm may be used
as a reference, if desired.
3.2 Sample Prior to use, antibody-containing samples should be clarified to

Clarification remove cellular debris and other contaminants as these can interfere
with assays. Clarification is also required to remove media compo-
nents which may interfere with the chromatographic process,
and an example of such a component is the commonly used pH
indicator, phenol red. Clarification steps are generally performed
directly before purification (see Note 7). In some cases, such as
when purifying recombinant protein expressed in bacteria, it is the
pellet which should be retained, rather than the supernatant. If
sample harvesting, clarification, and purification steps are not to
be performed in direct succession, the sample should be aliquoted
and stored at 20 C or 80 C to avoid any degradation or loss of
activity.
3.2.1 Clarification of 1. Centrifuge the sample in the range of 3,000–20,000 g for

Mammalian Cell Culture 30 min at 4 C.
(Hybridoma) 2. Decant the supernatant into fresh containers and discard pellet.
Proceed to protein precipitation, concentration or buffer
exchange to remove any remaining contaminants, for example,
phenol red and salts, and reduce sample volume (described in
Subheadings 3.3 and 3.4.3).
3.2.2 Clarification of 1. After collection of whole blood, leave the sample to coagulate
Serum at room temperature for up to 1 h.
2. Centrifuge sample for 10 min at 3,000 g and 4 C.
3. Decant the supernatant (serum) into a fresh container.
4. Filter serum through a 0.22 μm membrane filter and store
filtrate at 20 C for use in further purification steps.
3.2.3 Preparation of 1. Centrifuge culture at 3,000 g for 20 min at 4 C.

Bacterial Cultures 2. Discard supernatant and resuspend cell pellet in sonication/
equilibration buffer at 10 mL/1 g of pellet (see Note 8).
3. Separate resuspended cells into 50 mL tubes containing
5–10 mL sample per tube and leave on ice.
4. Sonicate the bacterial cells at 40% amplitude for 2 min (3 s
pulse, 2 s break). Ensure that samples remain on ice and that
sonication probe is submerged in cell suspension at all times.
5. Centrifuge cells in the range of 15,000–20,000 g for 30 min
at 4 C.
6. Filter supernatant through a 0.22 μm membrane filter to
remove any residual particulate matter and proceed to affinity
chromatography. If purification will not be performed immedi-
ately, store filtrate short-term at 20 C.
3.3 Reduction of Often the volume of the sample containing the antibody is unsuit-
Sample Volume able for application to bench-top chromatographic methods, for
instance, hybridoma cell culture media volumes. If the sample
volume is too large, methods such as precipitation or membrane
filtration may be applied. These allow for concentration of the
antibody within the sample, providing a smaller volume with
which to work. On occasion, precipitation, or concentration of
the antibodies in the sample may be sufficient for purification, and
no further chromatographic steps may be required. However, the
two methods used in conjunction offer an enhanced purification
compared to either step alone. This is important for later applica-
tions of the sample as the purity of the sample may affect parameters
such as antibody specificity or sensitivity. This section describes two
commonly used methods for reducing sample volume, ammonium
sulfate precipitation and membrane filtration.
3.3.1 Ammonium Sulfate 1. Add the clarified antibody sample to a beaker along with a
Precipitation stirrer bar. Note the volume of the sample.
2. Add saturated ammonium sulfate solution very slowly to the
sample until ammonium sulfate concentration reaches roughly
45–50%, that is, the volume of ammonium sulfate added is
equal to sample volume (see Note 9).
3. Leave beaker at 4 C for 4–6 h, or overnight.
4. Centrifuge precipitated mixture in the range of
3,000–10,000 g at room temperature for 30 min.
5. Pour off supernatant and leave pellet to air dry. This contains
the antibody.
6. For serum samples, resuspend the precipitate in PBS, roughly
30–50% of the original starting volume of the sample. For
hybridoma culture samples, resuspend pellet in 10% of the
starting volume with PBS.
7. Excess salt remaining in the sample may be removed via buffer
exchange (Subheading 3.4.3).
3.3.2 IgY Precipitation 1. Separate the egg yolk from the egg white, rinse the yolk in
ultrapure water, dry the yolk and drain/pipette the liquid yolk
into an appropriately tared receptacle.
2. Record the volume of the yolk in milliliters, assuming that 1 g is
equivalent to 1 mL.
3. Add five times the sample volume of cold delipidation reagent
under continuous gentle stirring and incubate this mixture at
4 C for 2–24 h.
4. After the incubation, remix the sample prior to centrifuging in
the range of 4,000–10,000 g for 15 min at 4 C.
5. Decant the supernatant into a fresh storage container and

add to it an equal volume of cold IgY Precipitation Reagent
with gentle mixing for 2 min.
6. Incubate the sample at 4 C from 1 h to overnight.
7. After the incubation, remix the sample and centrifuge in pre-
cooled tubes in the range of 4,000–10,000 g for 15 min at
4 C.
8. Discard the supernatant. The remaining pellet is considered
purified IgY and can be resuspended in a volume of PBS
equal to the starting volume of the original egg yolk. The IgY
can be stored at 20 C.
3.3.3 Sample 1. Add the appropriate molecular weight cutoff membrane to the
Concentration concentration/filtration system and pour in the sample.
2. Attach a pressure-source to the system and place on a magnetic
stirrer platform to begin concentration. Have a container ready
at the filtrate outlet to collect filtered liquid.
3. Filter sample until desired sample volume remains in the cell.
3.4 Purification of Protein A, G, A/G, L, and IMAC resins are among some of the
Antibodies Using most cost-effective and readily available resins; however, it is worth
Affinity noting that there are extensive affinity chromatography resin alter-
Chromatography natives, some of which may better suit the needs of the user.
Examples of these include anti-HA, anti-c-myc, amylose, anti-
FLAG, or glutathione resins. Investigation should be performed
prior to purification as to whether one of these less commonly used
resins may be more fit for purpose than the more widely applied
resins [38]. The choice of resin, and therefore ligand, is extremely
dependent on factors such as the species, class and structural format
of the antibody to be purified. The resins and protocols described in
the following sections are appropriate for the purification of a wide
range of antibody types at a relatively high level of purity. However,
it is pertinent to optimize parameters such as the wash, binding and
elution steps as the purity and antibody yield of the resultant
elution fractions may be improved by tailoring these. The following
purifications may be performed at the bench, either at 4 C or at
room temperature.
3.4.1 Protein A, G, and L 1. Equilibrate the resin and requisite buffers to room
Chromatography temperature.
2. Place a bottom cap on the column and add 2 mL of the relevant
resin.
3. Allow the resin to settle before removing the bottom cap and
draining storage solution. It is critical that the resin does not
run dry. Therefore, ensure the next step is performed as the
storage buffer meets the top of the resin bed, recapping the
column to do so, if necessary.
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whose mouths these artificial waterfalls are vomited. In this state, at
least, a jet d’eau forms but a disagreeable ornament in a garden,
which ought to unite every beauty of nature, and to disdain every
other.
The next object to St. Cloud, which attracted our attention, was
the famous manufactory of Sèvre. The beautiful porcelain, which
bears the name of this town, continues to be made here, under the
immediate protection of the government. We visited the shop, or
magazin, and were shown the several rooms of which it consists. In
all of these were tables, covered with specimens of china made
here, but I cannot say that they answered my expectation. They
were neither as various, nor as splendid, as one should suppose they
would be, at the principal dépôt of so renowned a manufactory.
Probably, the situation in which France has been during the
revolution, did not afford a sufficient number of purchasers, to
induce the managers to keep by them any considerable quantity of
expensive articles. There were several busts of Bonaparte in
different sizes, all of which were strikingly resembling. There were
also great and small busts of Voltaire, Franklin, and Rousseau.
Going thence to Versailles, we drove to Rambrand’s, which is
esteemed the principal hotel; but finding, on our arrival, that the
best rooms were engaged, we changed our plan, and proceeded to
le Petit Trianon in the park, which, formerly the much loved retreat
of Marie Antoinette, has, in the strange metamorphosis things as
well as men have experienced in France, become a common inn.
Having ordered dinner to be prepared in a small room, once
celebrated as the luxurious boudoir of the ill fated queen, we
proceeded to view the curiosities of Versailles. The park has lost
some trees, and has been neglected. In other respects, it is not
much altered. The orangerie[63] still retains, unimpaired, all its
beauty. We walked through long avenues of orange trees, all of
which are in high health and rich foliage. The gardener assured us,
that some of those which were of very large dimensions, had been
planted in the reign of Francis I.
We next visited the private library of the former kings of France,
situate in a separate house in the town. There is nothing very
particular in the building; but there were, above the several doors of
the library, extremely pretty paintings of the different capitals of
Europe. We were here shown a very beautiful collection of
illuminated paintings, representing the splendid fête and
tournaments given by the magnificent Lewis XIV.
Thence our guide wished to take us to the national manufactory of
fire arms, which is carried on with great activity in this town; but
having seen many acknowledgedly superior works of the same kind
in England, we declined visiting it, and proceeded at once to the
palace. This superb building has not suffered at all during the
revolution; though, from being neglected and uninhabited, it has
contracted a kind of gloom, which forcibly recals the misfortunes of
its last possessors, and the uncertainty of human grandeur. The
magnificent furniture, which the apartments once contained, has
been removed; but the walls are not without ornament, for the
palace having been made (probably with the view of preserving it
from popular violence) a musée central, or dépôt of the works of art,
now possesses several valuable pictures, and a few excellent
statues. Among the former, I remarked some good Claude Loraines,
and two beautiful portraits by Vincent. The subject of one was Henry
IV of France; and the other, that of the celebrated president, Molé.
The latter is painted in his parliamentary robes, heroically exposing
his breast to the violence of the mob, and doing his duty, unmoved
by the poniards raised against him. You seem to hear him exclaim,
as history records he did, “La distance est grande de la main d’un
assassin au cœur d’un honnête homme[64].”
We walked through the vast suite of rooms, which, once the seat
of gayety, splendour, luxury, and royal magnificence, are now the
abode of solitude, and the monument of fallen grandeur.
It is unnecessary to state the many reflections which this spot
created. We failed not to visit the apartment which the unfortunate
Lewis XVI occupied on the 6th of october, and in which Marie
Antoinette took refuge. We were also shown the balcony window
(now stopped up), where that virtuous and ill fated princess,
madame Elizabeth, with a magnanimity truly heroic, presented
herself, when the queen was called for, and being taken for her,
voluntarily subjected herself to all the brutal violence of an irritated
mob.
We likewise saw the opera house, built for the wedding of Lewis
XVI, when dauphin, and which, during the last reign, was sometimes
used as a theatre, and sometimes as a ball room. The apartment is
still perfect, but the scenes and decorations have been removed.
On leaving the palace, we visited several jets d’eau; but were
prevented from viewing the garden as particularly as we could have
wished, a violent shower of rain having overtaken us.
The waterworks and pleasure grounds appear to have been much
neglected.
We dined at the Little Trianon, and slept there. The room, which
fell to my share, was that which the unhappy Lewis formerly
occupied, and the key of the door had attached to it a label, on
which could still be discovered, though half effaced, the words,
“appartement du roi[65].”
In calling for our bill this morning, we found that this little inn (ci-
devant a royal residence) had two proprietors, one of whom lets the
apartments, and the other supplies the table in the character of
“traiteur.” With the charges of the latter we had no fault to find; but
the demand of the former was so ridiculously exorbitant, that have
kept the bill as a curiosity, of which I add the copy.
Petit Trianon logement[66].
Francs.
Trois appartemens de maître 36
Bougie 6
Bois 9
Quatre lits de domestique 12
Total 63
By way of reconciling us to this extravagant charge, the mistress

of the house sent her daughter to us, a very pretty girl, with the bill.
Our gallantry, however, did not subdue our reason, and we
determined to resist the demand. Our hostess having indignantly
refused the half, which we offered as amply sufficient, Mr. ⸺ and I
proceeded to Versailles, in pursuit of a juge de paix. After being sent
to two or three justices, who told us, that it was not within their
jurisdiction, we at last, in a miserable lodging, and at an obscure
house, found the magistrate of the division. His dress and his
appearance were not superiour to his residence, and from these
united circumstances, we were far from expecting that justice which,
in the result, we experienced.
Having heard our case, he granted a citation, requiring the
attendance of the landlord; and, of course, suspended his decision
till the arrival of the other party. While our servant, who carried the
summons, went to execute it, we were present at a curious trial, the
subject of which was a small quantity, I believe a quart, of vinegar.
The defendant was a coarse masculine woman, at least sixty years
of age, who, when she had exhausted all her fund of eloquence,
burst into tears, and talked of the weak unprotected stare of an
unhappy widow. The plaintiff was a dirty ill looking fellow, with a
witness of no better appearance. They all talked together; and the
justice, instead of being able to moderate their violence, found it
difficult to gain a hearing himself. After a wrangle of an hour, and
after swearing and counterswearing to the same fact, they went
away, without the business being finally settled.
What entertained me much, was, that these disputants, in the
middle of their harangues, turned round to my friend and me, and
seemed anxious, each in his turn, to convince us, by their
eloquence, of the justice of their respective cases; though we were
not only strangers to the business, but also to the laws on which this
important question was to be decided.
When our good landlady arrived, her bill was produced; and the
justice having declared how exorbitant he thought it, she justified
herself on three grounds.
1st. That we had not made a previous agreement; and ergo, that
she had a right to demand whatever she pleased.
2dly. That she paid a great rent “à la nation[67];” and that,
therefore, “la nation” ought to permit her to make her guests pay a
great rent for their lodgings.
3dly. That “l’ambassadeur de l’empereur Russe[68],” having lodged
at her house only a week before, and not having objected to a
charge of two louis per bed, “les milords anglois” ought to think her
present demand extremely reasonable.
Notwithstanding this very able defence, the justice told her, that
the law would not allow her d’écorcher les étrangers[69]; and very
equitably decreed, that we should pay 36 instead of 63 francs.
Madame received, very indignantly, the sum allotted her, and went
away in a rage, declaring that, in future, no person should sleep at
le petit Trianon, who would not bind himself before hand to pay the
price which “son excellence l’ambassadeur de toutes les Russies”
thought so reasonable.
So concluded our trial, which I have detailed as characteristic of
what is to be expected at inns in France, if prior arrangements be
not made by travellers; and likewise as an honourable proof, that
justice, though not clad in ermine, is fairly administered. In going
away, I was not a little surprised to find, that there were no costs to
pay, and that even the summons had been issued gratuitously.
In England, where we possess so admirable a system of laws, how
much are its advantages diminished, by the expenses attendant on
every process! for, as a distinguished public man once well observed,
though the temple of justice be open to all, it is like the London
tavern, only the favourites of fortune dare approach its threshold.
In returning to our inn, we passed by the royal stables, which are
still kept up, and filled with horses. These are now appropriated to
the use of the officers of the army, who come here to be instructed
in the menage, and who employ, for this purpose, the quondam
riding school of the king. The horses appeared, at least to an english
eye, very indifferent. We also saw here some arabians, lately arrived
from Egypt. They are extremely plain, lanky, and awkward; but the
groom assured us, on being asked if they were swift, “Oui, monsieur,
comme les oiseaux.” (“Yes, sir, as swift as birds.”) It was in vain to
object to outward form, when we learnt that these animals had the
talent of flying. If, according to the old jockey phrase, “no good
horse can have a bad colour,” certainly no horse who flies can be
ugly.
Before we left Versailles, we visited the garden of le petit Trianon,
which is rented by our honest landlady, and which may be seen, by
paying a small sum for a ticket at the gate. It is kept in tolerable
order, and has still strong marks of that good taste, with which it
was originally made. It is really, and not nominally, an english
garden; and would, even in our happy island, be deemed as prettily
laid out, as the smallness of its extent would permit.
The little theatre, built by the queen, situate within the precincts
of these grounds, is still in existence, and has suffered no loss,
excepting that of the beautiful glasses with which the boxes were
once splendidly illuminated. The last object, to which we were led at
Versailles, was “le grand Trianon,” that favourite spot of Lewis XVI.
This elegant building is also unhurt; and the fine marble pillars,
which form the entrance, excited all our admiration. The poverty,
into which the inhabitants of the town have fallen, in consequence of
the revolution, is strikingly apparent. In every corner, we were
surrounded by half-starved and half-naked beggars, whose
importunities were not a little troublesome.
In returning to Paris, we took the road of St. Germain. The old
castle still remains; but its outward appearance was so gloomy, that
we felt no inclination to visit the interiour. If the french monarch
intended to pay a compliment to the pretender, in giving him a
palace as nearly as possible resembling St. James’s, his choice was
admirable. The view from the terrace is pretty, but by no means
either as extensive, nor as rich, as I expected from its celebrity.
In continuing our road, we saw the celebrated waterworks of
Marly, which are preserved in all their perfection. We likewise passed
by the famous aqueduct, and by Malmaison, the private seat of the
first consul. The latter has nothing very particular to distinguish it. It
is simply a moderately sized house, situate near the river, but so low,
that it cannot command a very extensive prospect. I hear, the
grounds are well laid out, and that the furniture of the house unites
every thing which taste could order, or luxury afford. We reached
Paris about six o’clock; and my first employment, after dinner, has
been to write you this account, as I know that Versailles, and its
neighbourhood, are among the number of objects, about which
english curiosity is excited.
I am, &c.
LETTER XXV.
Long Champ, account of that annual promenade, date of its origin, and of
the great preparations made this year for attending it.—The bustle,
and gayety which it produced at Paris.
Paris, april the 16th, 1802, (27 germinal).
my dear sir,
All Paris has been alive for the last three days. Can you guess the
reason? Perhaps you will imagine, that the inhabitants, roused from
the state of lethargy, into which they have for some time back been
plunged, are beginning to give sincere but tardy marks of joy, at the
reestablishment of internal order, and external peace. Not at all, my
good friend. A subject, much more interesting to the parisians, is the
cause of the show and gayety so generally exhibited. Know, then,
that “Long Champ” has begun! I must now, like other learned
commentators, explain my explanation.
“Long Champ” is the name of a village, situate on the other side of
the “Bois de Boulogne,” of which latter place I spoke to you in a
former letter. In this village stood an abbey, or church; and one of
the holy fathers, some hundred years ago, had a voice of such
extraordinary sweetness, that, when high mass was performed,
crowds of Parisians flocked to hear him. His popularity was not
confined to the lower class, and the noblesse shared the curiosity of
the public. The fashion of going to Long Champ so rapidly increased,
that, in a short time, it was no uncommon thing to see whole strings
of splendid carriages at the door of the convent. The road to this
village became the favourite ride, and vanity soon discovered, that it
afforded an excellent opportunity of displaying all the varieties of
dress, and all the pomp of equipage. In the course of a few years, it
became an established custom, at this particular period of the year,
to make, during three days, not an humble pilgrimage, but a
splendid procession, to Long Champ. The mass and the singer were
soon forgotten; but the promenade continued, and increased every
year in the magnificence of parade. To appear, on this occasion, with
becoming grandeur, the haughty, but often distressed noble, would,
for months beforehand, deprive himself of his ordinary comforts. To
rival “les dames de bonne compagnie[70]” in richness of dress, in
show of equipage, and blaze of diamonds, was the grand object of
the admired belles of the opera house; and the means of doing so,
was the exacted price of those smiles, which the well beneficed
prelate, or the wealthy financier, were sometimes permitted to
enjoy. The Bourgeois and their wives appeared in their humble
cabriolets, but the former wore their Sunday apparel; and the latter
were loaded with all the tinsel finery, which, during the course of the
year, they had been able to collect. The common people, or la
canaille (as they were then indignantly called), were equally fond of
this procession; and, at the risk of being run over, crowded and
completed the show, some dressed in tattered regimentals, some in
faded silk coats, and ragged embroidered waistcoats, and others
with bag-wig’s and wooden shoes.
Such was the custom during “l’ancien régime.” The amusements of
the french vanished with their old political institutions, and
“horrendum dictu,” Long Champ was long unobserved.
Robespierre, and after him the directors, forbade every thing
which bore the least resemblance to the customs of former times;
but when Bonaparte came into power, the system was instantly
changed, and the people, left to follow their own inclinations,
greedily returned to all their former diversions. “Long Champ” was of
the number; and, since the 18th of brumaire, it has been gradually
recovering its ancient magnificence. This year, from the
reestablishment of peace, and the confluence of foreigners, it was
expected to be finer than ever; and vast preparations have, during
some weeks past, been making. Milliners tortured their fancy to
invent new fashions; mantuamakers passed whole nights without
sleep, in executing the orders which they had received; coachmakers
exerted themselves with all the art of their trade, and all the vanity
of their country, in endeavouring to imitate the carriages of the
english; horses were sent for from every part of the world;
regiments of tailors were employed in making coats for the beaux,
habits for the ladies, and laced jackets for their grooms; strings of
boots were seen dangling on the backs of porters in every quarter of
the town; saddles were as much in requisition, as if a great military
project, by the means of cavalry, had been in agitation; and I have
been confidently assured, that no less than three thousand pair of
leather breeches were ordered on the occasion.
In consequence of these active preparations, and of “Long
Champ” having been, for some weeks back, infinitely more the
subject of conversation than either the peace, or the reestablishment
of religion, I expected, at least, a very brilliant sight. I must say, I
was disappointed. The only thing which pleased me very much, was
the bustle which it produced in the town, and the gayety with which
it animated the faces of the Parisians. For three days, every vehicle
in the shape of a carriage, and every animal which claimed the name
of horse, has been dragged into use, and become part of the
procession. About two o’clock, a military guard was posted at the
beginning of the Champs Elisées, to preserve order, (for nothing
here is done without soldiers); and from that hour, till some time
after sunset, the crowd gradually increased. At three, the line of
carriages reached from “la place de la Concorde” to the “Bois de
Boulogne;” and, of course, there were frequent stoppages, even at
the beginning of the promenade. The road not employed in this
manner, was filled with equestrians of all ranks, and the walk on
both sides was equally thronged with passengers on foot. There
were some few elegant english equipages, well appointed, and
others spoiled, by the shabby appearance of the servants, or the
extreme badness of the horses. The french coachmakers, in one or
two instances, successfully imitated the fashions of London; but,
generally speaking, the attempt only served to prove the vast
distance which exists, between the two countries, in the art of
constructing carriages.
Mixed with “les voitures à l’anglaise, ou véritablement
anglaises[71]” were seen old fashioned berlins, family coaches, and
superannuated cabriolets of all descriptions. Phaetons, gigs,
curricles, and whiskies, completed the procession. Among the
horsemen were seen a few returned emigrants, who had so well
copied the dress of our young men of ton, that they might have
been mistaken for the beaux of Bond street; but the greater number
(malgré their leather breeches and boots, their blue frocks and high
crowned hats) betrayed the forgery, by the preposterous addition of
ear-rings, coloured capes, or pointed toes. The ladies appeared in
every variety of clothing. Some, who ventured to be their own
charioteers, assumed the neat and appropriate dress of an
“amazone,” or habit. Others, decorating, and concealing as little as
possible, the charms of their person, shone in all the brilliance of
their evening apparel. Worked gowns, laced caps, and showy
turbans, were sometimes exhibited from the windows of hackney
coaches; and a dirty buggy had, not unfrequently, the honour of
conveying three or four damsels, whose costume would not have
been unsuited to the first heroine of the stage. It is impossible to
describe, or convey, the faintest idea of the grotesque figures which
appeared on this occasion; and, notwithstanding the trouble and
expense to which so many individuals had exposed themselves, by
the purchase of new carriages, new liveries, new horses, new
dresses, and last, not least, new leather breeches, the whole
appeared to me but a shabby exhibition, dull amusement.
Moving, in slow procession, to the other side of the Bois de
Boulogne, during five or six hours, constituted the whole pleasure of
this vaunted fête. There were certainly some elegant carriages, and
some handsome horses; but the number was too inconsiderable to
make amends for the crowd of those of a contrary description.
Nothing could be more tiresome than sitting in one of these vehicles,
as they were compelled, every instant, to stop, on account of the
lengthened line, which increased every moment. Persons on
horseback were equally ill off, as it required the utmost care to avoid
being driven against the wheels of the carriages; and as for the
pedestrians, they were almost buried in a volley of dust.
Such is the celebrated promenade of Long Champ, which, though
an annual festival, appears to me a wretched and pitiful imitation of
Hyde park on an ordinary sunday. Yet the french are delighted with
their amusement; and in returning this evening, I heard on every
side, “Quel beau spectacle! quelles jolies voitures! quels magnifiques
chevaux! quelle belle parure! Vraiment c’est charmant[72]!”
It is not a little flattering to the vanity of an englishman, to see
how rapidly the french are adopting our fashions; and,
notwithstanding the awkward manner in which they are sometimes
copied, yet such is the general bias, that I entertain no doubt that,
in the space of ten years, (if the peace should last so long), it will
become almost impossible to distinguish, by his dress, a native of
France from one of England.
The ladies of Paris, and those of London, differ, indeed, very
widely in their toilet. Perhaps they might reciprocally improve by
observing each other; and while the former would do right to
respect and imitate the modesty, with which the latter are usually
clad, our fair countrywomen might also, without any injury to their
beauty, or any violation of that delicacy, which is their brightest
ornament, adopt some of that taste, elegance, and fancy, which are
often seen in the dress of a well bred frenchwoman.
Adieu, my dear sir. I am heartily tired of my subject, and fear you
will have been so some time. I therefore take my leave for the
present.
I am, &c.
LETTER XXVI.
Te Deum sung at Notre Dame, in honour of the peace and the
reestablishment of religion.—Military insolence.—Account of the
ceremony.—Illuminations in the evening.—Indifference of the people.
Paris, april the 18th, 1802, Easter Sunday (28 germinal.)
my dear sir,
To day will probably be long remembered in the annals of France, on

account of the promulgation of the law for (“l’établissement des
cultes”) the reestablishment of religion; on account of the definitive
treaty of peace with England, the ratifications of which were
exchanged this morning at the Thuilleries; and of the “Te Deum”
sung at Notre Dame, in honour of these united events.
I wished very much to be present at a ceremony, which was
rendered so particularly interesting by the number of curious
concurring circumstances, too obvious to be detailed. Having no
ticket, I went to the church at six o’clock in the morning, hoping to
make my way, among the crowd, into those places, which were not
appropriated to the constituted authorities. The doors were not
open; and about a hundred persons, who were already arrived,
stood enclosed in a kind of barrier, which seemed to have been put
up for the purpose of preventing too great a press at the first
opening of the gates. I placed myself against this bar, and hoped to
gain admittance in the second division. I was soon followed and
surrounded by a considerable crowd; and, after we had all remained
about two hours in this uncomfortable state, a detachment of
soldiers arrived, and attempted instantly to clear a passage. We
were already so squeezed together, that it was impossible to make
room for the military, without either losing our places, or incurring
the danger of suffocation. When the soldiers perceived that,
notwithstanding the blows which they dealt around them without
ceremony, the people did not immediately make way, they lost all
patience; and, not content with fixing their bayonets, called out for a
detachment of horse. The brandishing of the one, and the fear of
the other, soon dispersed the mob; but not till some had been
wounded, and several severely bruised.
I could not help reflecting, with some degree of indignation, on
this singular scene. In England, under a monarchical form of
government, the military are not allowed to interfere, but in cases of
positive danger, or actual insurrection; and even then under the
orders of a civil magistrate. In France, where the system is called
“republican,” and every man supposed to constitute a part of the
sovereignty, the body of the people, coming quietly to see the first
solemn service of that religion, which is said to be restored in
compliance with their wishes, are driven with blows and military
violence from the doors of that church, in which peace, liberty,
equality, and good order, are about to be celebrated. Perhaps,
indeed, it may be urged, that this was only a necessary precaution
of the police, and that the object of the guard was to prevent that
riot and danger to which the public, not so protected, would have
been exposed. The answer is plain. If it was thought necessary to
maintain order by the assistance of the military power, the sentinels
ought to have been placed the preceding night, or at the dawn of
morning. It was adding insult to cruelty, to permit the people to
assemble, and after the loss of several hours, and the endurance of
great fatigue, to dismiss them in the manner I have described.
It is needless for me to say, that I soon relinquished all hope of
getting into the church, and thought myself happy in being able to
make my escape unhurt from the claws of these heroes.
In going away, I perceived at the window of an adjoining hospital,
nearly opposite the church, some ladies of my acquaintance, who
were so obliging as to offer me a place near them, from which I
might see the procession.
I had scarcely taken this situation, when a ticket for one of the
privileged places in the church was given me by a person, who was
unwilling to risk the difficulties, with which the approach to the doors
seemed attended. After being sent about to different gates, I at last
found admittance at one. When I reached the gallery, it was so
completely full, that I found myself compelled to take refuge in the
orchestra. From this situation I was again driven by the soldiers; and
in despair I returned to the gallery, where, standing on the back of a
tottering chair, and with at least twenty rows of spectators before
me, I caught, not without some danger, a very imperfect glimpse of
this splendid ceremony.
What I did not see myself, I shall relate on the authority of
persons, who were more fortunately situate, and on whose accuracy
I know I can depend.
The procession began with a numerous escort of different
regiments. Among these were particularly remarked “les guides,” a
corps of handsome young men, clad in hussar dresses, and mounted
on beautiful horses, who excited universal admiration. Next to them
came the “gens d’armes,” or “régiment d’élites,” lately raised. They
are men of a very respectable appearance, in blue uniforms, faced
with yellow, whence long epaulets are suspended. These, as well as
the buttons, are of silver, as is the lace of their hats. Their horses are
black. The consular guards, and several regiments of the line,
completed the military cavalcade. The ministers of state, and the
“corps diplomatique,” came next, and formed a long line of
carriages. Those of the latter were drawn each by four horses, and
ornamented with all the escutcheons of heraldic pomp. Those of the
former were without arms; but they had all six horses, and their
servants, dressed alike, wore splendid liveries, now put on for the
first time, of yellow, gold, and red. A small corps of Mamalukes in
their egyptian costume, some of whom led unmounted arabians, and
a few aides-de-camp, immediately preceded the carriage, in which
sat Bonaparte, accompanied by the other two consuls. His coach,
new on the occasion, was simply elegant, and drawn by eight very
fine horses richly caparisoned. His servants appeared in green coats
and red waistcoats, on all the seams of which were rows of broad
gold lace. The consuls were received at the door of the church by
the archbishop of Paris, who placed over their head a dais (or
canopy).
Bonaparte, with Cambacères on his right, and le Brun on his left
hand, was conducted in this manner to a throne erected near the
altar, under which their three chairs were placed. A similar throne
appeared opposite, in which sat the cardinal legate.
The bishops bowed first to the altar, secondly to the consul, and
lastly to the cardinal. This was remarked by the public; as, under the
monarchy, the representative of the pope was permitted to receive
this homage before the sovereign of the country.
The oath settled by the concordat having been taken by the
clergy, high mass was instantly said.
At the conclusion of this ceremony, M. de Boisgelin, formerly
archbishop of Aix, lately named archbishop of Tours, ascended the
pulpit, and delivered a discourse appropriate to the occasion. I
regretted much, that the distance at which I was placed was so
great, that it was impossible for me to hear the venerable preacher,
who excited no little curiosity, from the singularity of his situation.
He is the same man, who, at the “sacre” or coronation, of Lewis XVI,
preached before that unfortunate monarch. His sermon will, no
doubt, be published in the “moniteur,” where you will have an
opportunity of seeing it.
It was the custom formerly on these occasions, for the bishop, in
beginning his discourse, to address himself to the king. A similar
form was observed to day, and the expression of “sire” was
exchanged for that of “citoyen premier consul.” After the sermon,
“Te Deum” was chanted. All the band of the opera house was
employed, and Lais and madame Bolla supplied the vocal parts. The
effect was fine, yet, comparatively, very inferiour to our musical
meetings in Westminster abbey. I heard some connoisseurs object to
the air, as not sufficiently grave or dignified for the subject which it
was intended to celebrate. As I am totally ignorant of music, I can
form no judgment as to the justice of the criticism.
The church was immensely full. The aisle was filled with the
military, the different uniforms of which had a splendid effect.
Behind the consuls sat the ambassadors, the ministers, and the
generals. In a box above, at the entrance of the chapel, was placed
madame Bonaparte, accompanied by her daughter and some other
ladies. On the other side was a similar box, appropriate to the use of
the ladies of the “corps diplomatique.”
The two galleries or choirs, which surround the church, were
divided into an orchestra for the music, seats for the different
constituted authorities, and places for such individuals as were
favoured with tickets. In the latter were of course seen all the
persons at Paris most distinguished for situation, talent, or beauty.
The coup d’œil altogether was very striking. The procession returned
with the same ceremony as that in which it arrived; and all the
streets of Paris were lined with spectators.
A discharge of sixty cannon was heard at the departure of the first
consul from the Thuilleries; and his arrival at the church, and his
return to the palace, were announced in the same manner.
In the evening, the palace was splendidly illuminated. Every
division of the arches forming the front towards the garden was
covered with lamps, and a lustre of lights was suspended from each.
The garden itself was prettily, but less brilliantly, decorated, than on
the fête in honour of the preliminaries.
All the public buildings and offices were also lighted; but the only
illumination at all remarkable, beside those which I have named, was
that of Mr. Jackson, his majesty’s envoy extraordinary. The gates of
“l’hôtel de Caramon,” where he lodges, were entirely covered with
lamps of different colours; the effect of which was much admired, as
at Paris that mode of decorating their rejoicings is unknown. On the
right hand were the letters R. F. (République Française); and on the
left, G. R. (Georgius Rex).
I forgot to mention that Bonaparte was much applauded by the
populace, in going to Notre Dame; and that madame received the
same compliment, though she went there without any parade, in a
plain handsome carriage, and seemed to decline, rather than to
court, the notice of the public.
During the illuminations there was no noise, and, indeed, no
expression of joy. Very few people were seen in the Thuilleries,
though the weather was fine, and the day sunday. The more I see of
the french, the more am I astonished and disgusted at the
indifference which they have contracted. Their dullness is the more
disagreeable, from it’s being unnatural; and I cannot help
exclaiming, every hour, with Voltaire,
Que je plains un françois, quand il est sans gaieté;

Loin de son élément le pauvre homme est jetté[73].
Adieu.
LETTER XXVII.
Palais de Justice.—Account of the different tribunals or courts of law.
Paris, april the 30th, 1802, (10 floréal.)
my dear sir,
I went this morning to the “Palais de Justice,” in order to visit the

different tribunals. The “façade,” or front, of this building has a
commanding appearance. A handsome iron railing, with three gates,
forms its barrier; after passing through which, you ascend a lofty
flight of stone steps. The avenues to the principal hall are filled with
shops of various descriptions, and particularly those of booksellers.
The hall, or central room, which is of considerable extent, forms a
kind of antichamber to the different courts. I went into one of the
“tribunals de premier instance,” in each of which three judges
preside. They wear long bands, and black coats, from which is
suspended a cloke or gown of black silk. The advocates plead in a
sort of bar; but, excepting being dressed in black, have no
distinguishing badge, or professional decoration. The judges had a
grave appearance; and, though they did not seem to be men of
much importance, conducted themselves with decency and
propriety. I was present while some causes were argued; but they
were not of sufficient consequence to enable me to form any
estimate of the talents of the advocates, now called, in the general
change of name, “des défenseurs officiaux[74].” As to their outward
garb, it was not prepossessing; and, if it were not unjust to form any
conclusion from mere exteriors, I should say, that a french counsel
and an english one appear to be drawn from a very different class of
society. I next saw a court, which, under the title of “tribunal de la
police correctionelle,” is charged with the investigation and
punishment of petty offences. I here heard the trial of a man
charged with pawning, for his own use, some goods belonging to a
shop, in which he was employed as a workman. The witnesses were
regularly examined; after which the criminal was very patiently
heard in his defence. As he had nothing to urge but his poverty and
the charges of a large family, he was found guilty by the judges, (for
I observed no jury) and was sentenced, though an old offender, to
only six months imprisonment. This trial having satisfied my curiosity
about “la police correctionelle,” I next visited the chief or supreme
court of the republic, which is called “le tribunal de cassation.” Here
every thing bore a more dignified appearance. The room was lofty,
the seats elevated, and the judges (whose number was
considerable) seemed, by their dress, their manner, and their
language, to be well suited to the important functions of their office.
They wore black and red gowns, with cocked hats, the cords of
which were of gold lace. Nothing can be more respectable than the
exterior of this court; of the proceedings of which I could form no
idea, as the judges were employed in reading papers relating to
mere matters of form. I imagine, that this is the tribunal intended, in
some respects, to replace the parliament of Paris. The magistrates,
as far as I could form an opinion from this cursory visit, seem men
of education, learning, and polished manners.
Before I left the Palais de Justice, I looked in at the criminal court
of the “départment de la Seine.” A culprit was reading a long written
defence, which I had not the patience to hear concluded. The room
was handsome, and the proceedings orderly and correct. I saw here
nothing like a jury; yet I am told, that all capital offences are tried
by that mode of process. An Italian was a few days ago tried in this
court, and convicted of assassination. I regret much, that I was not
present at the trial. I did not hear of the circumstance till to day. He
yesterday underwent the punishment of the guillotine, being led to
the scaffold in a red shirt, this disgrace being added to the sentence
in cases of murder. I ought to mention, in honour of the present
criminal laws of France, that this is the first individual, who has been
condemned to death, during the six months which I have passed at
Paris.
Underneath the “Palais de Justice” is situate that fatal prison,
called “la Conciergerie.” It was here that the sanguinary Robespierre
immured the daily victims of his wild and unrelenting tyranny; who
awaited, within its dismal walls, the signal of death, under the
insulting and degraded name of trial. It was here, that rank, beauty,
age, philosophy, virtue, and patriotism, took the places of vice; and,
in the caverns destined to receive the blackest perpetrators of
hideous crimes, were hurled, among multitudes of other innocent
and dignified characters, the learned Condorcet, the ingenious
Lavoisier, the respectable Madame Roland, the venerable
Malesherbes, and the lovely, courageous, and once haughty queen
of France. When I have been forced to make such painful reflections,
in viewing the different objects which present themselves at Paris, I
have always found some consolation in looking round me, and
seeing how completely that system of suspicion, bloodshed, and
injustice, has passed away. Those horrors, so disgraceful to France,
took place in a moment of national delirium (if I may be permitted
the expression); and the inhabitants of Paris, who committed, or
rather suffered, the scenes of judicial murder, which every day
contaminated the streets of the capital, now, restored to their
senses, are the first to deplore and execrate them. I am persuaded,
that crimes like these can never again find their way into the
polished metropolis of this great, brave, and ingenious people.
With this hope I shall conclude my letter—a hope, in which I am
certain that you will warmly and cordially unite.
I am, &c.
LETTER XXVIII.
The gardens and walks of Paris.
Paris, may the 2d, 1802 (12 floréal).
my dear sir,
Whenever you come to Paris, come with the smiling month of may.
On my arrival here, at the end of october, I was disgusted with the
dirt of the streets, the mire of the Thuilleries, the ruts of the
Boulevards, and the general gloom of the town. Accustomed to take
a great deal of exercise, I could not persuade myself to be shut up,
the whole of every day, either in a hot room, or a close carriage. I
therefore continued to walk about: but, while my feet were cut to
pieces at every step, I was frequently in danger of being run over by
a rapid cabriolet, or squeezed to atoms under the ponderous wheels
of an overloaded cart. Nor was I consoled for this hazardous
undertaking by meeting with any conversable persons of my
acquaintance.
The parisians, who have carriages, never think of walking during
the severe days of winter; and those who do not possess that
convenience, spend the greater part of every day at home. To save
the expense of a fiacre[75], they will, indeed, sometimes use their
feet in going to a restaurateur’s, a spectacle, or a ball, or in paying
some of those innumerable visits, in which an inhabitant of this town
passes half his life; but, as to taking exercise for health, it never
enters into the calculations of a frenchman. Nothing, therefore,
could be so dismal as the streets in the months of november,
december, and january; and a severer punishment could not be
devised for the daily murder of time committed by our Bond street
loungers, than to condemn them to a three months pilgrimage, at
that period of the year, round the streets of Paris.
The spring has, with fine weather, changed the face of every thing
here; and a person fond of exercise may now have all the
advantages which he can possibly desire.
The Thuilleries and Champs Elisées, which in winter are almost
impassable, now offer excellent gravel walks, and delightful shade
under the long avenues of lofty trees. Here crowds are collected at
almost every hour of the day; and, besides long lines of pedestrians,
rows of chairs are filled with ladies eating ice, and politicians reading
newspapers.
The Bois de Boulogne affords an admirable ride for persons in
carriages or on horseback, and a lengthened walk for those on foot.
Besides these, there are several delightful gardens open to the
public in different parts of the town. The Boulevards, which surround
Paris on every side, are now seen to great advantage.
The walks are in high order, the trees are in rich foliage; and the
number of mountebanks, printsellers, quack doctors, and shows of
all kinds, collected here, and the crowds of persons and carriages
which are constantly passing, make them present a very curious and
lively scene.
The “Hameau de Chantilly,” or Elisée de Bourbon, very near the
Champs Elisées, has a very pretty, though not an extensive garden,
into which, for a few sols, you gain admittance. It was hence I saw
madame Garnerin ascend, about a month ago, in a balloon,
unaccompanied by any one. The day was fine; and we were all
much delighted with the courage of the fair heroine, who was the
first female that had ventured alone on such an expedition.
She descended a few leagues from Paris, and supped, the same
evening, in public, at the Hameau, where she was received with
universal and merited applause.
I am particularly pleased with a garden, called “Mousseux, ou les
délices de Chartre,” situate in the Fauxbourg St. Honoré, and within
the gates of Paris. It formerly belonged to the duke of Orleans; but,
having been confiscated with the rest of his enormous fortune, is
now national property, and open every day for the use of the public.
The garden is arranged in the english taste, commands an extensive
view, and has all the advantages and appearance of grounds at a
considerable distance from a capital. Fine verdure, trees of every
kind in the must luxuriant blossom, variety of flowers, a clear sky,
and birds warbling a thousand wild notes, make you entirely forget
the town; and the whole seems a fairy scene produced by
enchantment.
The only things in bad taste, I mean the modern antiques and
drawbridges, are now falling into decay; and the artificial ruins are
daily becoming real ones.
The salon or house consists of a long gallery; but it has been so
much neglected during the revolution, that it is now in a very
tottering and dangerous state. “Mousseux” is still a most delightful
spot, and must have been exquisitely so, when kept in proper order.
I doubt much, if such a garden is to be found within the walls of any
other capital in Europe.
I ought to mention, that, though the town is completely hidden in
the shady walks of this charming retreat, one of the best views of
Paris is enjoyed from a hillock of easy ascent, situate in the centre of
these grounds. There is a traiteur at the door of the garden, where
dinners may be ordered; but his accommodations (as far as can be
judged from the outward appearance of his habitation) I should
suppose not very superiour. I have heard, however, of large and
fashionable parties, who have dined here. Nothing is paid for walking
in the garden.
At the further end of the Bois de Boulogne, about two miles from
Paris, there is another place of this kind, which, whenever you come
to Paris, I recommend your visiting. It is called “Bagatelle,” and
formerly belonged to the Comte D’Artois, who is said to have built
the house and arranged the grounds in the space of six weeks. The
building, which is now an inn or tavern, is light and elegant; and the
garden (allowing for some few exceptions) is laid out with
considerable taste. Near the house there is a very pretty and very
striking view of the bridge of Neuilly. The accommodations here are
good, and a person fond of the country cannot pass a fine evening
more agreeably, than by dining at Bagatelle, and strolling afterwards
about the grounds.
Before I conclude my account of the gardens of Paris, I ought to
mention two, which, are opened at this season of the year, at a late
hour, and usually frequented after the opera, or other spectacles; I
mean Frescati and Tivoli.
Frescati consists of a large house and small garden, situate on the
Boulevard. The gate stands in the corner of “la rue de la Loi.” The
apartments, elegantly painted with italian landscapes, are large and
numerous, and splendidly lighted every evening. The garden was
illuminated last night, for the first time this season, and is as pretty
as its limited extent can permit. It is the fashion to come here about
ten o’clock; and the amusement consists in walking about, chatting
with your friends, eating ices and cakes, or drinking tea, punch, or
lemonade, the sale of which articles constitute the whole profits of
the landlord, to whom nothing is paid for admittance. Frescati is, in
short, a kind of coffee house; and, notwithstanding the smell of
brandy, gin, and rum, generally prevalent, is frequented by ladies as
well as gentlemen. When I first came here, I supposed that these
ladies were of a certain description: but I was soon undeceived; and,
besides seeing at this place the most respectable families of Paris,
was assured by a ci-devant comtesse excessively rigid on matters of
etiquette, “que toute la bonne compagnie y alloit[76].” After this
authoritative decision, it would be presumptuous to doubt the
propriety of going to Frescati; and our most scrupulous
countrywomen may, without apprehension of being taken either for
“filles” or “parvenues,” enjoy this strange and singular amusement.
Tivoli is but just opened for the season. I have been there once. It
is a large and beautiful garden, situate in “la rue St. Lazare,” in the
“Chaussée d’Antin.” It was illuminated with much taste; the trees are
lofty; and the whole seemed to resemble what I imagine our
Vauxhall was, before it was covered in.
There was a band of music, and dancing, on a platform erected
for the purpose. There was also a party of tumblers. The company
was not either very numerous or very genteel; but the night was not
hot, and fine weather is necessary to render this place agreeable; as
there is no house or shelter of any kind. In the months of July or
august Tivoli must be delightful.
I forgot to mention, that there were roundabouts, (as they are
vulgarly called in England) on which full grown people were very
gravely amusing themselves. I heard, the other day, of a duel, which
took place in consequence of a dispute for one of these places. You
will scarcely believe the report; yet I am every day convinced, that
there is nothing so ridiculous, that fancy can suppose, which does
not actually and frequently take place in this most extraordinary
town.
Having given you this short sketch of the amusements “al fresco,”
I shall conclude with repeating my recommendations to you,
whenever you come to Paris, to come in the spring. Winter is,
indeed, the time for private society; but I have found from
experience, that a foreigner has so little to expect on this head, that
it is much wiser to choose a season, when an infinite variety of
amusements, and all the charms of Nature, in their richest and
happiest colours, offer a sure and constant fund of pleasure.
I am, &c.
LETTER XXIX.
The manufactory of “Gobelins,” the observatory, “les Enfans trouvés,”
“Champ de Mars,” les Invalides, and the temple of Mars, containing
the colours taken from different nations, and the tomb of Turenne.—
Le Musée des Monumens françois, or collection of monuments.—List
of the most esteemed of these.—Note to this letter contains the
account of a dinner at the first consul’s.
Paris, may the 5th, 1802 (15 floréal).
my dear sir,
As my stay at Paris draws towards a conclusion, I have occupied the

three or four last days in visiting those objects of curiosity, which as
yet I had neglected to see.
The manufactory of Gobelins deserves all its celebrity. The colours,
the design, and the execution of the tapestry made here, are equal
to the productions of the finest painting. I was shown some
specimens, which were uncommonly beautiful, particularly two
pieces, one of which represented the assassination of the admiral
Coligni, and the other the heroic conduct of the président Molé,
copied from the picture at Versailles, an account of which I have
already given.
There are ninety persons now employed, of whom I saw several at
work. It is astonishing with what facility they seem to perform the
most difficult tasks, but I am told that the art is not learnt without
much time and considerable attention. The apprenticeship requires
six years, and at least eighteen are necessary to make a proficient.
The workmen are not locked up within the walls of the manufactory,
as was the case during the monarchy, but they are kept under the
constant “surveillance[77] of the police.” Most of the pieces now in
hand have been ordered by the first consul, and are destined to
form the ornament of St. Cloud, and other public buildings.
From the Gobelins, situate in the most distant part of the
Fauxbourg St. Germain, I drove along the new Boulevard to the
observatory. I found here only some common sized telescopes, on
which I observed with pride the respectable name of “Dollond,” of
London. I was informed that a magnificent instrument of this kind is
preparing on the plan of Herschel, which is to be twenty-two feet
long, with a speculum of platina. It is to be moved on a platform, for
the purpose of making observations, by means of a machine
invented for the purpose. I ascended the top of the building, and the
view thence, which commands all Paris, is grand and striking.
Near the observatory is the nursery of that humane establishment
called “les Enfans trouvés,” which is still kept up on the old
philanthropic plan. Orphan children, deprived by death of their
parents, or abandoned by them, are received here without question,
recommendation, or inquiry, and are nursed with tenderness, well
fed, properly educated, and lastly, qualified for some trade or
profession, in which they are afterwards placed at the expense of
the public. Their infancy is passed in the building shown to me; they
are, at a certain age, sent into the country, for the benefit of the air,
and then return to the principal hospital of the institution at Paris,
where their education is completed. Their number is seldom less
than a thousand.
I shall continue to speak of the different objects I have lately
seen, in the same order in which I visited them.
The Champ de Mars, where, on the 14th of july, 1790, I was
present, when the unfortunate Lewis XVI received and repeated the
oath of fidelity to that constitution which was so soon violated, has
still the remains of that vast amphitheatre, made by the activity and
zeal of the parisians in the course of fourteen days, and on which
were seated nearly a million of people. I recollected all the spots,
where the principal authorities were placed on that memorable day;
and it will be needless for me to repeat the innumerable reflections
which were created by a remembrance of the extraordinary and
many-coloured events which have since occurred. The École
Militaire, which is now a barrack for the consular horse guards,
forms the front and principal ornament of the Champ de Mars, which
is terminated on the other side by the river Seine. L’École Militaire
was built, in 1751, from a plan of Gabriel. It has a handsome façade,
and a lofty dome, with a dial, and the figures of Time and
Astronomy.
The building of “les Invalides” presents one of the most striking
objects of Paris. Besides the beauty of its construction, its handsome
entrance, its four courts, its celebrated clock, its lofty dome, and
elegant pillars, it contains, in the principal hall, or chapel, now called
“le Temple de Mars,” the colours, or ensigns, taken during the war,
by the republican armies, from the different powers opposed to
France. This beautiful room at least a hundred feet long, is lined on
all sides with the badges of triumph, many of which bear, from their
tattered appearance, the most convincing proofs of not having been
obtained without considerable difficulty. Among the innumerable
colours of all nations, I perceived, with pride, that there were only
two or three english; and these, from their size, had belonged to
some ship, perhaps to a merchantman, or to that man of war, which,
after sharing the glories of Aboukir, was taken by five french vessels
of the line, after a resistance no less honourable than her former
success. In the centre of this spacious apartment, to the right,
surrounded by the trophies of his successors and countrymen, is
placed the tomb of Turenne. This monument was removed to the
Temple of Mars by the present government, and placed here, with
considerable pomp, after having been saved from the fury of
jacobinical rage, and preserved, by the care of le Noir, in the “Musée
des Monuments françois.”
The circumstance which I have just mentioned, naturally leads me
to speak of the last named institution, which I visited yesterday for
the second time. On my arrival at Paris, I went to see le Musée des
Monuments françois; and not being as much struck with the
exhibition as I expected, from its great renown, I purposely
postponed speaking of it, till I had had an opportunity of examining
it again with all the attention it deserved. After several hours
employed in this second view, I continue of my former opinion, that
the spot[78], in which these monuments are collected, is infinitely
too small; that the garden, meant to be the tranquil site of
sepulchral honours, and the calm retreat of departed grandeur, is on
so limited a scale, is so surrounded with adjoining houses, and
altogether so ill arranged, that, instead of presenting the model of
“Those deep solitudes ...

Where heav’nly pensive Contemplation dwells,
And ever musing Melancholy reigns,”
it might easily be mistaken for the working yard of a statuary, or the

pleasure ground of a tasteless citizen, decked out with Cupids,
Mercuries, and Fauns.
It must, however, be acknowledged, that “le Noir,” by whose care
this establishment was formed, deserves great credit for the courage
with which he rescued the many precious monuments here
deposited, from the barbarous and undiscriminating fury of
revolutionary vandalism, for the perseverance and attention which
has marked his conduct in the arrangement, and for the plan
(whether successful or not, I shall not pretend to examine) of
presenting complete and exact representations of the art of building
in the different ages. Among the innumerable tombs, crowded
together in this collection, of which a catalogue, in large octavo, is
sold at the door, I observed many of great beauty, taste, and
symmetry. Kings, poets, belles, philosophers, and painters, torn from
the graves, in which, for centuries past, their remains had tranquilly
reposed, would have had no stone to record their past celebrity, if
this institution had not existed. The arts, too, are highly indebted to
the founder, for the specimens of sculpture, many of them chefs
d’œuvre, which are here seen in all their original perfection. Models,
too, of ancient and celtic buildings are added to those of french
architecture.
Le Noir’s favourite plan of having a chapel for each century,
ornamented with all the appropriate decorations, and containing the
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Affinity Modification Of Biopolymers First Edition Knorre

Cyclodextrins in Chromatography Rsc Chromatography Monos

Advances In Chromatography Volume 44 Advances in

Lingua Latina A College Companion Based on Hans Ørberg s

■■■■■■■ ■■■■ ■■■■■■■■■■■■ ■■■■■■■■ ■ ■■■■■ ■■■■■■■■■

The Book of Kubernetes Alan Hohn

Sound and Sense in Classical Arabic Poetry 1st Edition

Cardiothoracic Surgery 2nd Edition Joanna Chikwe

For further volumes:

Methods and Protocols

ISSN 1064-3745 ISSN 1940-6029 (electronic)

© Springer Science+Business Media, LLC, part of Springer Nature 2022

Affinity chromatography is a versatile biochemical separation technique which relies on

Houston, TX, USA B. Vijayalakshmi Ayyar

PART I APPLICATIONS OF AFFINITY CHROMATOGRAPHY

PART II LIGAND SELECTION AND AFFINITY MATRIX

12 Ligand Selection for Affinity Chromatography Using Phage Display . . . . . . . . . . 159

SEVGI ASLIYÜCE • Department of Chemistry, Hacettepe University, Ankara, Turkey

MARKUS NIEHAUS • Department of Molecular Nutrition and Biochemistry of Plants, Leibniz

Applications of Affinity Chromatography

Antibody Purification Using Affinity Chromatography

Key words Affinity purification, Antibodies, Chromatography, Methods, Recombinant proteins

Antibodies are members of the immunoglobulin family. These

Previously, antibodies were largely isolated from the body fluids

Pre-Purification Affinity Post-Purification

Application Binding Washing Elution

Variable Light Constant Light Variable Heavy Constant Heavy

similar fashion to the monoclonal antibody. However, develop-

widely used method is inappropriate for many of the recombinant

1.2 Antibody When purifying monoclonal antibodies, knowledge of the subclass

1.3 Sample As aforementioned, antibodies are found in a range of sources,

and G was developed. Termed protein A/G, this fusion protein

1.5.2 Immobilized Metal In contrast to the previously described affinity chromatographic

Chemicals can be sourced from Sigma-Aldrich, unless otherwise

2.1.2 ELISA 1. Commercial isotyping ELISA kits, for example, Invitrogen’s Ig

2.2 Sample 1. Centrifuge.

2.2.2 Clarification of 1. Centrifuge.

2.2.3 Clarification of 1. Centrifuge.

2.3 Reduction of 1. Saturated ammonium sulfate solution (see Note 2).

2.3.2 IgY Purification 1. Centrifuge.

2.3.3 Sample 1. Concentration system, for example, Amicon® Stirred Ultrafil-

2.4 Purification of 1. Polypropylene disposable columns, 20 mL capacity (Bio-Rad).

2.4.2 Immobilized Metal 1. Polypropylene disposable columns, 20 mL capacity.

2.4.3 Buffer Exchange 1. Centrifuge.

3.1 Antibody As previously mentioned, characterization of the antibodies within

Isotyping is not as pertinent when purifying recombinant antibo-

3.2 Sample Prior to use, antibody-containing samples should be clarified to

3.2.1 Clarification of 1. Centrifuge the sample in the range of 3,000–20,000 g for

3.2.3 Preparation of 1. Centrifuge culture at 3,000 g for 20 min at 4 C.

5. Decant the supernatant into a fresh storage container and

Petit Trianon logement[66].

By way of reconciling us to this extravagant charge, the mistress

Paris, april the 16th, 1802, (27 germinal).

Paris, april the 18th, 1802, Easter Sunday (28 germinal.)

To day will probably be long remembered in the annals of France, on

3.2.3 Preparation of 1. Centrifuge culture at 3,000 g for 20 min at 4 C.