-Features contributions from experienced scientists with hands-on expertise in the field presenting step-by-step methodologies, tips, and associated explanatory notes-Includes a large section on the generation and purification of recombinant proteins-Addresses protein biochemistry with clear, step-by-step techniques
A prerequisite for elucidating the structure and function of any protein is the prior purification of that protein. This necessity has led to the development of many purification schemes and chromatographic methods for the isolation of native proteins from complex sources. In Protein Chromatography: Methods and Protocols, leading researchers present clear protocol-style chapters that are suitable for newcomers and experts alike. The book opens with vital topics in protein biochemistry, addressing such areas as protein stability and storage, avoiding proteolysis during chromatography, protein quantitation methods including immuno-qPCR, and the contrasting challenges that microfluidics and scale-up production pose to the investigator, and then it segues into key methods involving the generation and purification of recombinant proteins through recombinant antibody production and the tagging of proteins, amongst other means, as well as many variations on classic techniques such as ion-exchange and immunoaffinity chromatography. Written in the highly successful Methods in Molecular Biology™ series format, protocols chapters include introductions to their respective subjects, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls.Authoritative and up-to-date, Protein Chromatography: Methods and Protocols will greatly aid scientists in establishing these essential techniques in their own laboratories and furthering our understanding of the many imperative functions of proteins.
Part I: Overviews1. A Digest of Protein PurificationDermot Walls, Robert McGrath, and Sinéad T. Loughran2. Gel-Filtration ChromatographyCiarán Ó´Fágáin, Philip M. Cummins, and Brendan F. O´Connor3. Immunoaffinity ChromatographyJenny Fitzgerald, Paul Leonard, Elaine Darcy, and Richard O´Kennedy4. Avoiding Proteolysis During Protein ChromatographyBarry J. Ryan5. Scale-Up of Protein Purification: Downstream Processing IssuesJohn Joseph Milne6. Phage Display: A Powerful Technology for the Generation of High Specificity Affinity Reagents from Alternative Immune SourcesWilliam J.J. Finlay, Laird Bloom, and Orla Cunningham7. Engineering Protein StabilityCiarán Ó´Fágáin8. Microfluidics in Protein ChromatographyFrank A. Gomez9. Tagging Recombinant Proteins to Enhance Solubility and Aid PurificationDermot Walls and Sinéad T. LoughranPart II: Protocols10. Storage and Lyophilization of Pure ProteinsCiarán Ó´Fágáin11. Differential Precipitation and Solubilization of ProteinsBarry J. Ryan12. Ion-Exchange Chromatography: Basic Principles and Application to the Partial Purification of Soluble Mammalian Prolyl OligopeptidasePhilip M. Cumminsa, Oonagh Dowling, and Brendan F. O´Connor13. Protein Quantitation and Analysis of PurityEva M. Campion, Sinéad T. Loughran, and Dermot Walls14. Purification of Proteins Fused to Glutathione S-TranferaseSandra Harper and David W. Speicher15. Purification of Proteins Fused to Maltose-Binding ProteinMario Lebendiker and Tsafi Danieli16. Purification of Proteins from Baculovirus-Infected Insect CellsLuke O´Shaughnessy and Sean Doyle17. Purification of Poly-Histidine-Tagged ProteinsSinéad T. Loughran and Dermot Walls18. Immobilized Metal Affinity Chromatography/Reversed-Phase Enrichment of Phosphopeptides and Analysis by CID/ETD Tandem Mass SpectrometryRosana Navajas, Alberto Paradela, and Juan Pablo Albar19. Tag Removal by Site-Specific Cleavage of Recombinant Fusion ProteinsAdam Charlton and Michael Zachariou20. Purification of Antibodies Using Affinity ChromatographyElaine Darcy, Paul Leonard, Jenny Fitzgerald, Martin Danaher, and Richard O´ Kennedy21. Optimized Generation of High-Affinity, High-Specificity Single-Chain Fv Antibodies from Multi-Antigen Immunized ChickensWilliam J.J. Finlay, Laird Bloom, and Orla Cunningham22. Measuring Protein-Protein Interactions Using BiacorePaul Leonard, Stephen Hearty, and Richard O´Kennedy23. Ultra-Performance Liquid Chromatography-Mass Spectrometry of ProteinsTimothy R. Croley24. Hydrophobic Interaction ChromatographyPhilip Cummins and Brendan O´Connor25. Fast Protein Liquid ChromatographyAshkan Madadlou, Siobhan O´Sullivan, and David Sheehan26. Shotgun Proteomics: A Qualitative Approach Applying Isoelectric Focusing on Immobilized pH Gradient and LC-MS/MSLaurent Geiser, Ali R. Vaezzadeh, Jacques M. P. Deshusses, and Denis F. Hochstrasser27. Shotgun Proteomics: A Relative Quantitative Approach Using Off-Gel Electrophoresis and LC-MS/MSLaurent Geiser, Loïc Dayon, Ali R. Vaezzadeh, and Denis F. Hochstrasser28. Clinical Proteomics: Liquid Chromatography-Mass Spectrometry (LC/MS) Purification SystemsMichael Henry and Paula Meleady29. Strategies for the Purification of Membrane ProteinsSinead Marian Smith30. A Multi-Step Chromatographic Strategy to Purify Three Fungal Endo-?-GlucanasesTracey McCarthy and Maria G. Tuohy
This book examines vital topics in protein biochemistry and presents methods involving the generation and purification of recombinant proteins through recombinant antibody production and the tagging of proteins, as well as variations on classic techniques.
A prerequisite for elucidating the structure and function of any protein is the prior purification of that protein. This necessity has led to the development of many purification schemes and chromatographic methods for the isolation of native proteins from complex sources. In Protein Chromatography: Methods and Protocols, leading researchers present clear protocol-style chapters that are suitable for newcomers and experts alike. The book opens with vital topics in protein biochemistry, addressing such areas as protein stability and storage, avoiding proteolysis during chromatography, protein quantitation methods including immuno-qPCR, and the contrasting challenges that microfluidics and scale-up production pose to the investigator, and then it segues into key methods involving the generation and purification of recombinant proteins through recombinant antibody production and the tagging of proteins, amongst other means, as well as many variations on classic techniques such as ion-exchange and immunoaffinity chromatography. Written in the highly successful Methods in Molecular Biology(TM) series format, protocols chapters include introductions to their respective subjects, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls.Authoritative and up-to-date, Protein Chromatography: Methods and Protocols will greatly aid scientists in establishing these essential techniques in their own laboratories and furthering our understanding of the many imperative functions of proteins.
A Digest of Protein Purification.- Gel-Filtration Chromatography.- Immunoaffinity Chromatography.- Avoiding Proteolysis During Protein Chromatography.- Scale-Up of Protein Purification: Downstream Processing Issues.- Phage Display: A Powerful Technology for the Generation of High Specificity Affinity Reagents from Alternative Immune Sources.- Engineering Protein Stability.- Microfluidics in Protein Chromatography.- Tagging Recombinant Proteins to Enhance Solubility and Aid Purification.- Storage and Lyophilization of Pure Proteins.- Differential Precipitation and Solubilization of Proteins.- Ion-Exchange Chromatography: Basic Principles and Application to the Partial Purification of Soluble Mammalian Prolyl Oligopeptidase.- Protein Quantitation and Analysis of Purity.- Purification of Proteins Fused to Glutathione S-Tranferase.- Purification of Proteins Fused to Maltose-Binding Protein.- Purification of Proteins from Baculovirus-Infected Insect Cells.- Purification of Poly-Histidine-Tagged Proteins.- Immobilized Metal Affinity Chromatography/Reversed-Phase Enrichment of Phosphopeptides and Analysis by CID/ETD Tandem Mass Spectrometry.- Tag Removal by Site-Specific Cleavage of Recombinant Fusion Proteins.- Purification of Antibodies Using Affinity Chromatography.- Optimized Generation of High-Affinity, High-Specificity Single-Chain Fv Antibodies from Multi-Antigen Immunized Chickens.- Measuring Protein-Protein Interactions Using Biacore.- Ultra-Performance Liquid Chromatography-Mass Spectrometry of Proteins.- Hydrophobic Interaction Chromatography.- Fast Protein Liquid Chromatography.- Shotgun Proteomics: A Qualitative Approach Applying Isoelectric Focusing on Immobilized pH Gradient and LC-MS/MS.- Shotgun Proteomics: A Relative Quantitative Approach Using Off-Gel Electrophoresis and LC-MS/MS.- Clinical Proteomics: Liquid Chromatography-Mass Spectrometry (LC/MS) Purification Systems.- Strategies for the Purification of Membrane Proteins.- A Multi-Step ChromatographicStrategy to Purify Three Fungal Endo-beta-Glucanases.
Inhaltsverzeichnis
Part I: Overviews
1. A Digest of Protein Purification
Dermot Walls, Robert McGrath, and Sinéad T. Loughran
2. Gel-Filtration Chromatography
Ciarán Ó¿Fágáin, Philip M. Cummins, and Brendan F. O¿Connor
3. Immunoaffinity Chromatography
Jenny Fitzgerald, Paul Leonard, Elaine Darcy, and Richard O¿Kennedy
4. Avoiding Proteolysis During Protein Chromatography
Barry J. Ryan
5. Scale-Up of Protein Purification: Downstream Processing Issues
John Joseph Milne
6. Phage Display: A Powerful Technology for the Generation of High Specificity Affinity Reagents from Alternative Immune Sources
William J.J. Finlay, Laird Bloom, and Orla Cunningham
7. Engineering Protein Stability
Ciarán Ó¿Fágáin
8. Microfluidics in Protein Chromatography
Frank A. Gomez
9. Tagging Recombinant Proteins to Enhance Solubility and Aid Purification
Dermot Walls and Sinéad T. Loughran
Part II: Protocols
10. Storage and Lyophilization of Pure Proteins
Ciarán Ó¿Fágáin
11. Differential Precipitation and Solubilization of Proteins
Barry J. Ryan
12. Ion-Exchange Chromatography: Basic Principles and Application to the Partial Purification of Soluble Mammalian Prolyl Oligopeptidase
Philip M. Cumminsa, Oonagh Dowling, and Brendan F. O¿Connor
13. Protein Quantitation and Analysis of Purity
Eva M. Campion, Sinéad T. Loughran, and Dermot Walls
14. Purification of Proteins Fused to Glutathione S-Tranferase
Sandra Harper and David W. Speicher
15. Purification of Proteins Fused to Maltose-Binding Protein
Mario Lebendiker and Tsafi Danieli
16. Purification of Proteins from Baculovirus-Infected Insect Cells
Luke O¿Shaughnessy and Sean Doyle
17. Purification of Poly-Histidine-Tagged Proteins
Sinéad T. Loughran and Dermot Walls
18. Immobilized Metal Affinity Chromatography/Reversed-Phase Enrichment of Phosphopeptides and Analysis by CID/ETD Tandem Mass Spectrometry
Rosana Navajas, Alberto Paradela, and Juan Pablo Albar
19. Tag Removal by Site-Specific Cleavage of Recombinant Fusion Proteins
Adam Charlton and Michael Zachariou
20. Purification of Antibodies Using Affinity Chromatography
Elaine Darcy, Paul Leonard, Jenny Fitzgerald, Martin Danaher, and Richard O¿ Kennedy
21. Optimized Generation of High-Affinity, High-Specificity Single-Chain Fv Antibodies from Multi-Antigen Immunized Chickens
William J.J. Finlay, Laird Bloom, and Orla Cunningham
22. Measuring Protein-Protein Interactions Using Biacore
Paul Leonard, Stephen Hearty, and Richard O¿Kennedy
23. Ultra-Performance Liquid Chromatography-Mass Spectrometry of Proteins
Timothy R. Croley
24. Hydrophobic Interaction Chromatography
Philip Cummins and Brendan O¿Connor
25. Fast Protein Liquid Chromatography
Ashkan Madadlou, Siobhan O¿Sullivan, and David Sheehan
26. Shotgun Proteomics: A Qualitative Approach Applying Isoelectric Focusing on Immobilized pH Gradient and LC-MS/MS
Laurent Geiser, Ali R. Vaezzadeh, Jacques M. P. Deshusses, and Denis F. Hochstrasser
27. Shotgun Proteomics: A Relative Quantitative Approach Using Off-Gel Electrophoresis and LC-MS/MS
Laurent Geiser, Loïc Dayon, Ali R. Vaezzadeh, and Denis F. Hochstrasser
28. Clinical Proteomics: Liquid Chromatography-Mass Spectrometry (LC/MS) Purification Systems
Michael Henry and Paula Meleady
29. Strategies for the Purification of Membrane Proteins
Sinead Marian Smith
30. A Multi-Step Chromatographic Strategy to Purify Three Fungal Endo-ß-Glucanases
Tracey McCarthy and Maria G. Tuohy
Klappentext
A prerequisite for elucidating the structure and function of any protein is the prior purification of that protein. This necessity has led to the development of many purification schemes and chromatographic methods for the isolation of native proteins from complex sources. In Protein Chromatography: Methods and Protocols, leading researchers present clear protocol-style chapters that are suitable for newcomers and experts alike. The book opens with vital topics in protein biochemistry, addressing such areas as protein stability and storage, avoiding proteolysis during chromatography, protein quantitation methods including immuno-qPCR, and the contrasting challenges that microfluidics and scale-up production pose to the investigator, and then it segues into key methods involving the generation and purification of recombinant proteins through recombinant antibody production and the tagging of proteins, amongst other means, as well as many variations on classic techniques such as ion-exchange and immunoaffinity chromatography. Written in the highly successful Methods in Molecular Biology¿ series format, protocols chapters include introductions to their respective subjects, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls.nnAuthoritative and up-to-date, Protein Chromatography: Methods and Protocols will greatly aid scientists in establishing these essential techniques in their own laboratories and furthering our understanding of the many imperative functions of proteins.
Features contributions from experienced scientists with hands-on expertise in the field presenting step-by-step methodologies, tips, and associated explanatory notes
Includes a large section on the generation and purification of recombinant proteins
Addresses protein biochemistry with clear, step-by-step techniques
Includes supplementary material: sn.pub/extras
