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Antibody Purification – Handbook
Antibody
Purification
Handbook
18-1037-46
Edition AC
www.chromatography.amershambiosciences.com
Percoll
Methodology and Applications
18-1115-69
Ficoll-Paque Plus
For
in vitro
isolation of lymphocytes
18-1152-69
GST Gene Fusion System
Handbook
18-1157-58
2-D Electrophoresis
using immobilized pH gradients
Principles and Methods
80-6429-60
Antibody Purification
Handbook
18-1037-46
The Recombinant Protein Handbook
Protein Amplification and Simple Purification
18-1142-75
Protein Purification
Handbook
18-1132-29
Ion Exchange Chromatography
Principles and Methods
18-1114-21
Affinity Chromatography
Principles and Methods
18-1022-29
Hydrophobic Interaction
Chromatography
Principles and Methods
18-1020-90
Gel Filtration
Principles and Methods
18-1022-18
Reversed Phase
Chromatography
Principles and Methods
18-1134-16
Expanded Bed Adsorption
Principles and Methods
18-1124-26
Chromatofocusing
with Polybuffer and PBE
18-1009-07
Microcarrier cell culture
Principles and Methods
18-1140-62
Handbooks
from Amersham Biosciences
MabSelect, Hybond, HiTrap, GSTrap, Sephadex, Superdex, HisTrap, Sepharose, MAbTrap, ÄKTA, FPLC,
HiPrep, PhastSystem, PhastGel, SOURCE, RESOURCE, HiLoad, STREAMLINE, ECL, ECL Plus, MicroSpin,
Mono Q, Mono S, Monobeads, BioProcess, GSTPrep, BioDirectory, Tricorn and Drop Design are trademarks
of Amersham Biosciences Limited.
Amersham and Amersham Biosciences are trademarks of Amersham plc.
BIACORE is a trademark of Biacore AB.
Multipipette and Eppendorf are trademarks of Eppendorf-Netheler-Hinz GmbH.
Tween is a trademark of ICI Americas Inc.
Cibacron is a registered trademark of Ciba-Geigy Corp.
Coomassie is a trademark of ICI plc.
Triton is a trademark of Union Carbide Chemicals and Plastics Co.
All goods and services are sold subject to the terms and conditions of sale of the company within the
Amersham Biosciences group that supplies them. A copy of these terms and conditions is available on request.
© Amersham Biosciences AB 2002 – All rights reserved.
Amersham Biosciences AB, Björkgatan 30, SE-751 84 Uppsala, Sweden
Amersham Biosciences UK Limited, Amersham Place, Little Chalfont, Buckinghamshire HP7 9NA, England
Amersham Biosciences Corp., 800 Centennial Avenue, PO Box 1327, Piscataway NJ 08855, USA
Amersham Biosciences Europe GmbH, Munzinger Strasse 9, D-79111 Freiburg, Germany
Amersham Biosciences K.K., Sanken Bldg. 3-25-1, Hyakunincho Amersham Shinjuku-ku, Tokyo 169-0073, Japan
1
Antibody Purification
Handbook
2
Contents
Introduction ............................................................................................................. 5
Chapter 1
Antibody structure, classification and production ...................................................... 7
Native sources ............................................................................................................ 7
Genetically engineered sources ................................................................................... 10
Chapter 2
Sample preparation................................................................................................ 13
Sources and their associated contaminants .................................................................. 13
Extraction of recombinant antibodies and antibody fragments ........................................ 13
Clarification of serum, ascitic fluid, culture supernatant or cell lysates ........................... 15
Sample preparation before purification ........................................................................ 16
Chapter 3
Simple, rapid purification by affinity chromatography .............................................. 27
IgG, IgG classes, fragments and subclasses ................................................................. 29
Using Protein G Sepharose media ............................................................................... 30
Using MAbTrap Kit .................................................................................................... 33
Using Sepharose media coupled to native or recombinant protein A ................................ 35
Fab, F(ab')
2
fragments ............................................................................................... 38
IgA .......................................................................................................................... 40
IgD .......................................................................................................................... 40
IgE .......................................................................................................................... 40
IgM .......................................................................................................................... 40
Avian IgY from egg yolk .............................................................................................. 43
Making immunospecific purification media .................................................................. 45
Chapter 4
Immunoprecipitation .............................................................................................. 51
Chapter 5
Multi-step purification strategies ............................................................................ 55
Selection and combination of purification techniques ................................................... 56
Selection of media for multi-step purification ............................................................... 60
Examples of multi-step purification ............................................................................. 62
Chapter 6
Removal of specific contaminants after initial purification ....................................... 71
Bovine immunoglobulins ............................................................................................ 71
Albumin and transferrin ............................................................................................. 72
a2-macroglobulin and haptoglobulin ........................................................................... 74
Dimers and aggregates ............................................................................................... 74
DNA and endotoxins .................................................................................................. 75
Affinity ligands .......................................................................................................... 75
3
Chapter 7
Large-scale purification ......................................................................................... 77
Considerations for monoclonal antibody purification ...................................................... 78
Combining sample preparation and capture for Fc-containing antibodies ......................... 79
BioProcess Media for production ................................................................................. 80
Custom Designed Media and Columns ......................................................................... 80
Appendix 1 ............................................................................................................ 81
Analytical assays during purification ............................................................................ 81
Appendix 2 ............................................................................................................ 83
Selection of purification equipment ............................................................................. 83
Appendix 3 ............................................................................................................ 84
General instructions for affinity purification with HiTrap columns ................................... 84
Appendix 4 ............................................................................................................ 86
Column packing and preparation ................................................................................. 86
Appendix 5 ............................................................................................................ 89
Storage of biological samples ..................................................................................... 89
Appendix 6 ............................................................................................................ 90
Table of amino acids .................................................................................................. 90
Appendix 7 ............................................................................................................ 92
Converting from linear flow (cm/hour) to volumetric
flow rates (ml/min) and vice versa ............................................................................... 92
Appendix 8 ............................................................................................................ 93
Conversion data: proteins, column pressures ................................................................ 93
Appendix 9 ............................................................................................................ 94
Principles and standard conditions for purification techniques ....................................... 94
Product index ...................................................................................................... 101
Additional reading ............................................................................................... 102
References .......................................................................................................... 103
Ordering information ............................................................................................ 104
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