Protein Chromatography Methods and Protocols

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.

Table of contents

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-β-Glucanases Tracey McCarthy and Maria G. Tuohy

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