Protein Protein Purification D Handbook Purification Handbook Charge Back to Collection Amersham 18-1132-29 Biosciences Edition AC
Protein Purification Handbook 18-1132-29 Edition AC Protein Purification Ð Handbook
Handbooks from Amersham Pharmacia Biotech Antibody Purificatio 1g103246 The recombinant protein handbook Protein Purification 183229 lon Exchange Chromatography Methods Affinity Chromatography Metods Chromatofocusing d Methods Gel Filtration Microcarrier cell culture
Additional reading and reference material Antibody Purification Code No. 18-1037-46 Recombinant Protein Handbook Code No. 18-1142-75 Gel Filtration Principles and Methods Code No. 18-1022-18 Ion Exchange Chromatography Principles and Methods Code No. 18-1114-21 Hydrophobic Interaction Chromatography Principles and Methods Code No. 18-1020-90 Affinity Chromatography Principles and Methods Code No. 18-1022-29 Expanded Bed Adsorption Principles and Methods Code No. 18-1124-26 Gel Filtration Columns and Media Selection Guide Code No. 18-1124-19 Ion Exchange Columns and Media Selection Guide Code No. 18-1127-31 HIC Columns and Media Product Profile Code No. 18-1100-98 Affinity Chromatography Columns and Media Product Profile Code No. 18-1121-86 Sample Clean-up, Proteins and Peptides Code No. 18-1128-62 Convenient Protein Purification - HiTrap™ Column Guide Code No. 18-1129-81 Protein and Peptide Purification Technique Selection Code No. 18-1128-63 Protein Purification - major techniques poster Code No. 18-1123-93 Protein Purification - strategies poster Code No. 18-1129-75 Protein Purification, Principles, High Resolution Methods and Applications, J-C. Janson and L. Rydén, 1998, 2nd ed. Wiley VCH Code No. 18-1128-68 Handbook of Process Chromatography, G.Sofer and L.Hagel, 1997, Academic Press Code No. 18-1121-56 Protein Purification, Principles and Practice, R.K. Scopes. 1994, Springer Advanced Texts in Chemistry Ed. Springer Verlag New York Inc. HiTrap, Sepharose, STREAMLINE, Sephadex, MonoBeads, Mono Q, Mono S, MiniBeads, RESOURCE, SOURCE, Superdex, Superose, HisTrap, HiLoad, HiPrep, INdEX, BPG, BioProcess, FineLINE, MabTrap, MAbAssistant, Multiphor, FPLC, PhastSystem and ÄKTA are trademarks of Amersham Pharmacia Biotech Limited. Amersham is a trademark of Amersham plc. Pharmacia and Drop Design are trademarks of Pharmacia Corporation. Coamatic is a trademark of Chromogenix AB. Coomassie is a trademark of ICI plc. Triton is a trademark of Union Carbide Chemicals and Plastics Co. Tween is a trademark of ICI Americas Inc. All goods and services are sold subject to the terms and conditions of sale of the company within the Amersham Pharmacia Biotech group that supplies them. A copy of these terms and conditions is available on request. © Amersham Pharmacia Biotech AB 2001 – All rights reserved. Amersham Pharmacia Biotech AB Björkgatan 30, SE-751 84 Uppsala, Sweden Amersham Pharmacia Biotech UK Limited Amersham Place, Little Chalfont, Buckinghamshire HP7 9NA, England Amersham Pharmacia Biotech Inc 800 Centennial Avenue, PO Box 1327, Piscataway, NJ 08855 USA Amersham Pharmacia Biotech Europe GmbH Munzinger Strasse 9, D-79111 Freiburg, Germany Amersham Pharmacia Biotech KK, Sanken Bldg. 3-25-1, Hyakunincho, Shinjuku-ku, Tokyo 169-0073, Japan 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 Handbooks from Amersham Pharmacia Biotech 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
Protein Purification Handbook
Protein Purification Handbook
Contents Introduction. Chapter 1 Purification Strategies-A Simple Approach 7 Three Phase Purification Strategy 10 Chapter 2 Preparation Before You Start. Sample Extraction and Clarification. 14 Chapter 3 Three Phase Purification Strategy .17 Principles 17 Selection and Combination of Purification Techniques Sample Conditioning. 24 Chapter 4 Capture. .27 Chapter 5 Intermediate Purification 35 n6 38 Chapter 7 Examples of Protein Purification Strategies. .43 Three step purification of a recombinant enzyme 43 Three step purification of a recombinant antigen binding fragment .47 Two step purification of a monoclonal antibody. 52 One step purification of an integral membrane protein. 55 Chapter 8 Stora Conditions . 59 Extraction and Clarification Chapter 9 Principles and Standard Conditions for Purification Techniques . .71 Ion exchange (IEX). .71 Hydrophobic interaction (HIC). .77 Affinity (AC). 83 Gel filtration (GF). .86 90 93
Contents Introduction.5 Chapter 1 Purification Strategies - A Simple Approach .7 Preparation .8 Three Phase Purification Strategy .8 General Guidelines for Protein Purification .10 Chapter 2 Preparation .11 Before You Start . 11 Sample Extraction and Clarification .14 Chapter 3 Three Phase Purification Strategy .17 Principles .17 Selection and Combination of Purification Techniques .18 Sample Conditioning .24 Chapter 4 Capture .27 Chapter 5 Intermediate Purification .35 Chapter 6 Polishing .38 Chapter 7 Examples of Protein Purification Strategies .43 Three step purification of a recombinant enzyme .43 Three step purification of a recombinant antigen binding fragment .47 Two step purification of a monoclonal antibody .52 One step purification of an integral membrane protein .55 Chapter 8 Storage Conditions .59 Extraction and Clarification Procedures. 60 Chapter 9 Principles and Standard Conditions for Purification Techniques .71 Ion exchange (IEX) .71 Hydrophobic interaction (HIC) .77 Affinity (AC) .83 Gel filtration (GF) .86 Reversed phase (RPC) .90 Expanded bed adsorption (EBA) .93
Introduction d6oguisite otechno logy. provides ice and examples purification.Protein purifica ion varies from simple one-st tep precipitation procedures to large scale validated production processes.Often more than one purification step is necessary to reach the desired purity.The key to successful and efficient protein purification is to select the most appropriate techniques, optimise their performance to suit the requirements and combine them in a logical way to maximise yield and minimise the number of steps required. Most purification schemes involve some form of chromatography.As a result chromatography has become an essential tool in every laboratory where protein purification is needed.Different chromatography techniques with different selecti vities can form werful combinations for the purification of any biomolecule The develo nbinant dna techni ues has nised the tion of proteins in l quant rg sare often 中只 nts are still present and there myappert beer of me a few simple guidelines and application of the Three Phase Purification Strategy the process can be planned and performed simply and easily,with only a basic knowledge of the details of chromatography techniques. Advice codes: general advice for any purification advice for large scale purification e advice for micro scale purification shortcuts advice on media selection 5
5 Introduction The development of techniques and methods for protein purification has been an essential pre-requisite for many of the advancements made in biotechnology. This handbook provides advice and examples for a smooth path to protein purification. Protein purification varies from simple one-step precipitation procedures to large scale validated production processes. Often more than one purification step is necessary to reach the desired purity. The key to successful and efficient protein purification is to select the most appropriate techniques, optimise their performance to suit the requirements and combine them in a logical way to maximise yield and minimise the number of steps required. Most purification schemes involve some form of chromatography. As a result chromatography has become an essential tool in every laboratory where protein purification is needed. Different chromatography techniques with different selectivities can form powerful combinations for the purification of any biomolecule. The development of recombinant DNA techniques has revolutionised the production of proteins in large quantities. Recombinant proteins are often produced in forms which facilitate their subsequent chromatographic purification. However, this has not removed all challenges. Host contaminants are still present and problems related to solubility, structural integrity and biological activity can still exist. Although there may appear to be a great number of parameters to consider, with a few simple guidelines and application of the Three Phase Purification Strategy the process can be planned and performed simply and easily, with only a basic knowledge of the details of chromatography techniques. Advice codes: general advice for any purification advice for large scale purification advice for micro scale purification shortcuts advice on media selection