Gespeichert in:
| Titel: | Using antibodies a laboratory manual |
|---|---|
| Von: |
Ed Harlow ; David Lane
|
| Person: |
Harlow, Ed
Verfasser aut Lane, David |
| Hauptverfassende: | , |
| Format: | Buch |
| Sprache: | Englisch |
| Veröffentlicht: |
Cold Spring Harbor, NY
CSHL Press
1999
|
| Schlagworte: | |
| Online-Zugang: | http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=008390382&sequence=000002&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
| Beschreibung: | XIV, 495 S. Ill., graph. Darst. 9 Taf. |
| ISBN: | 0879695447 0879695439 9780879695446 |
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| 300 | |a XIV, 495 S. |b Ill., graph. Darst. |e 9 Taf. | ||
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| 650 | 4 | |a Anticorps - Manuels de laboratoire | |
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| 650 | 4 | |a Antibodies |x isolation & purification |v Laboratory Manuals | |
| 650 | 4 | |a Immunochemistry |v Laboratory Manuals | |
| 650 | 4 | |a Immunochemistry |v Laboratory manuals | |
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| adam_text | Antibody Structure
and Function
Antibodies are host
proteins produced in
response to the presence
of foreign molecules,
organisms, or other
agents in the body.
Antibodies are
synthesized
predominantly by
plasma cells, a
terminally differentiated
cell of the B-lymphocyte
lineage, and circulate
throughout the blood
and lymph, where they
bind to the antigens.
Once formed, the
antibody-antigen
complexes tire removed
from circulation mostly
through phagocytosis by
macrophages.
This
antibody response is one
of the key mechanisms
that a host organism uses
to protect against the
action oftorcign
molecules or
1
Antibody-Antigen
Interactions
/V/t interaction of an
antibody with an
antigen forms the basis
of all immunochemical
techniques. This
chapter discusses the
properties of the
antibody—antigen
interaction and is
divided into three
sections. The first
summarizes the
structure of the
antibody-antigen
bonds; the second
covers the strength of
these interactions, a
characteristic known as
affinity; and the third
presents the factors that
contribute to
tlie
overall stability of
immune complexes, a
property called avidity.
21
3
Choosing
Antibodies
Every
imniunochetnical
technique presents the
antigen in a different
physical context.
Immunostaining
displays the antigen
immobilized in its
native, but complex,
cellular context;
inununoprecipitations
present the antigen in
solution surrounded
by huge numbers of
other contaminating
molecules;
immunoblotting leaves
the antigen fully
denatured and
partially purified but
bound to a solid
support. Not
surprisingly, given this
diversity of antigen
displays, each
immuociienucal
technique requires
antibodies with
different properties.
Handling
Antibodies
This chapter presents
techniques that are
recommended to store,
purify, and label
antibodies. In general,
these techniques are
sufficient for handling
antibodies that are
made in your or your
colleagues labs. Most
commercial suppliers
who provide antibodies,
either labeled or
unlabeled, provide
suggestions on how to
store, handle, and use
their antibody
products.
61
39
Theory
Staining Ceils
Staining Tissues
7
1
mm unoprecipitation I mmunoblottinq
Immunostaining can
be used to pinpoint the
suhcellular localization
of a protein antigen, to
follow its changing
cellular address as cells
respond to stimuli, or
to compare its locale to
other proteins in the
same cell. With careful
controls, you may also
be able to get an idea of
how much protein is
present in the cell. In
this book, the
immunostaining
procedures have been
separated into two
major variations based
on the source of the
cells to be examined. In
this chapter, staining
cells growing in tissue
culture is discussed.
101
Immunostaining of
tissues of whole organisms
can be used to examine
the localization of
antigens in physiological
settings. Using these
methods, you can follow
an antigen s distribution
during development,
mark the location of a
particular cell type in a
multicellular in vivo
setting, or determine the
presence of an antigen in
a diseased tissue. The
protocols normally
require multiple steps
over several days as well
as extensive knowledge of
the architecture of the
tissues being studied.
These procedures demand
methods to preserve the
structure of tissues, which
unfortunately are often
damaging to the antigens.
Immunoprecipitation
allows the partial
purification of
antigens, normally
proteins, from complex
mixtures of soluble
molecules. The
antigens can be
purified up to 10,000-
fold by simple and
rapid methods that
collect the proteins on
inert beads. The
technique takes about
a day and can be
combined with any
other method that can
utilize immobilized
proteins as a starting
material.
221
151
Immunoblotting
provides a reliable
method to check any
sample for the presence
of protein antigen. The
assay identifies the
protein on the basis of
both its interaction
with a specific antibody
and its relative
molecular weight.
Immunoblotting can be
used to determine other
characteristics of your
antigen, such as the
relative abundance of
the antigen or
association with other
well-characterized
antigens. It is also a
useful method to
characterize new
antibody preparations.
Immunoblotting
involves multiple
simple steps that take
about a day to
complete.
267
Practice
-лл/
9
Immunoafŕimty
Purification
Immunoaffinity
purification takes
advantage of the high
binding affinity and
specificity of an
antibody for its antigen
to allow large
quantities of antigen to
be isolated in native or
near-native states. Not
all antibodies are
suitable for
immunoaffinity
purifications, but when
a good antibody is
available, the
procedure is quick and
reliable. The procedure
can be scaled to any
size, takes not more
than a half-day to
perform, and can
achieve levels of
purification
unmatched by other
methods of
chrot
η
a tograp hy.
311
10
Tagging Proteins
Proteins can be tagged using
recombinant
DNA
techniques to allow their
ready purification and their
specific detection in
different host cell systems.
These include tags designed
to help specifically with
detection (such as green
fluorescent protein tags) or
with purification (such as
the His tags and
glutathione-S-transferase
tags). Epitope tags
—
short
peptide
sequences to which
strong and specific
antibodies have already
been produced
—
can be
used for detection and
purification using all of the
immunological methods
described in this book. This
chapter describes the
different available tag
systems, their advantages
and disadvantages, and
how to choose the right tag
for your experiment.
345
Practice
11
Epitope Mapping
Determining the
binding sites (epitopes)
for monoclonal and
polyclonal antigens on
protein antigens often
provides extremely
useful information
that greatly extends
the power of
immunochemical
analysis. Epitope
mapping is used to
examine the specificity
of the immune
response or to
distinguish between
different antibodies.
379
Appendixes
Appendix I contains
information about
Electrophoresis;
Protein Techniques are
covered in Appendix
II; and Appendix III
provides General
Information.
409
Useful InFo
|
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| dewey-search | 616.07/98 |
| dewey-sort | 3616.07 298 |
| dewey-tens | 610 - Medicine and health |
| discipline | Chemie / Pharmazie Biologie Medizin |
| format | Book |
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| id | DE-604.BV012371857 |
| illustrated | Illustrated |
| indexdate | 2024-12-20T10:29:04Z |
| institution | BVB |
| isbn | 0879695447 0879695439 9780879695446 |
| language | English |
| oai_aleph_id | oai:aleph.bib-bvb.de:BVB01-008390382 |
| oclc_num | 246090931 |
| open_access_boolean | |
| owner | DE-29 DE-29T DE-20 DE-19 DE-BY-UBM DE-355 DE-BY-UBR DE-703 DE-91G DE-BY-TUM DE-526 DE-83 DE-634 |
| owner_facet | DE-29 DE-29T DE-20 DE-19 DE-BY-UBM DE-355 DE-BY-UBR DE-703 DE-91G DE-BY-TUM DE-526 DE-83 DE-634 |
| physical | XIV, 495 S. Ill., graph. Darst. 9 Taf. |
| publishDate | 1999 |
| publishDateSearch | 1999 |
| publishDateSort | 1999 |
| publisher | CSHL Press |
| record_format | marc |
| spellingShingle | Harlow, Ed Lane, David Using antibodies a laboratory manual Anticorps - Manuels de laboratoire Immunochimie - Manuels de laboratoire Laboratoriumonderzoek gtt Monoklonale antistoffen gtt Wetenschappelijke technieken gtt Wissenschaftliches Arbeiten Antibodies isolation & purification Laboratory Manuals Immunochemistry Laboratory Manuals Immunochemistry Laboratory manuals Immunoglobulins Laboratory manuals Antikörper (DE-588)4002290-0 gnd Immuncytochemie (DE-588)4161368-5 gnd Methode (DE-588)4038971-6 gnd |
| subject_GND | (DE-588)4002290-0 (DE-588)4161368-5 (DE-588)4038971-6 |
| title | Using antibodies a laboratory manual |
| title_auth | Using antibodies a laboratory manual |
| title_exact_search | Using antibodies a laboratory manual |
| title_full | Using antibodies a laboratory manual Ed Harlow ; David Lane |
| title_fullStr | Using antibodies a laboratory manual Ed Harlow ; David Lane |
| title_full_unstemmed | Using antibodies a laboratory manual Ed Harlow ; David Lane |
| title_short | Using antibodies |
| title_sort | using antibodies a laboratory manual |
| title_sub | a laboratory manual |
| topic | Anticorps - Manuels de laboratoire Immunochimie - Manuels de laboratoire Laboratoriumonderzoek gtt Monoklonale antistoffen gtt Wetenschappelijke technieken gtt Wissenschaftliches Arbeiten Antibodies isolation & purification Laboratory Manuals Immunochemistry Laboratory Manuals Immunochemistry Laboratory manuals Immunoglobulins Laboratory manuals Antikörper (DE-588)4002290-0 gnd Immuncytochemie (DE-588)4161368-5 gnd Methode (DE-588)4038971-6 gnd |
| topic_facet | Anticorps - Manuels de laboratoire Immunochimie - Manuels de laboratoire Laboratoriumonderzoek Monoklonale antistoffen Wetenschappelijke technieken Wissenschaftliches Arbeiten Antibodies isolation & purification Laboratory Manuals Immunochemistry Laboratory Manuals Immunochemistry Laboratory manuals Immunoglobulins Laboratory manuals Antikörper Immuncytochemie Methode |
| url | http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=008390382&sequence=000002&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
| work_keys_str_mv | AT harlowed usingantibodiesalaboratorymanual AT lanedavid usingantibodiesalaboratorymanual |
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