Guide to Protein Purification, Second Edition provides a complete update to existing methods in the field, reflecting the enormous advances made in the last two decades. In particular, proteomics, mass spectrometry, and DNA technology have revolutionized the field since the first edition's publication but through all of the advancements, the purification of proteins is still an indispensable first step in understanding their function. This volume examines the most reliable, robust methods for researchers in biochemistry, molecular and cell biology, genetics, pharmacology and biotechnology and sets a standard for best practices in the field. It relates how these traditional and new cutting-edge methods connect to the explosive advancements in the field. This "Guide to" gives imminently practical advice to avoid costly mistakes in choosing a method and brings in perspective from the premier researchers while presents a comprehensive overview of the field today.
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Table of Contents
1. Why Purify Enzymes?
Section I: Developing Purification Procedures 2. Strategies and Considerations for Protein Purifications 3. Use of Bioinformatics in Planning a Protein Purification 4. Preparing A Purification Summary Table
Section II: General Methods for Handling Proteins and Enzymes 5. Setting Up A Laboratory 6. Buffers: Principle and Practice 7. Measurement of Enzyme Activity 8. Quantitation of Protein 9. Concentration of Proteins and Removal of Solutes 10. Maintaining Protein Stability
Section III: Recombinant Protein Expression and Purification 11. Selecting an Appropriate Method for Expressing a Recombinant Protein 12. Bacterial Systems for Production of Heterologous Proteins 13. Expression in the Yeast Pichia Pastoris 14. Baculovirus-Insect Cell Expression Systems 15. Recombinant Protein Production By Transient Gene Transfer Into Mammalian Cells 16. Tagging For Protein Expression 17. Refolding of Solubilized Inclusion Body Proteins
Section IV: Preparation of Extracts and Subcellular Fractionation 18. Advances In Preparation of Biological Extracts for Protein Purification 19. Isolation of Subcellular Organelles and Structures
Section V: Purification Procedures: Bulk Methods 20. Protein Precipitation Techniques 21. Affi-Gel Blue for Nucleic Acid Removal and Early Enrichment of Nucleotide Binding Protein
Section VI: Purification Procedures: Chromatographic Methods 22. Ion Exchange Chromatography 23. Gel Filtration 24. Protein Chromatography on Hydroxyapatite Columns 25. Theory and Use of Hydrophobic Interaction Chromatography in Protein Purification Applications
Section VII: Purification Procedures: Affininty Methods 26. Affinity Chromatography: General Methods 27. Immobilized-Metal Affinity Chromatography (IMAC) 28. Identification, Production, and Use of Polyol-Responsive Monoclonal Antibodies for Immunoaffinity Chromatography
Section VIII: Purification Procedures: Electrophoretic Methods 29. One-Dimensional Gel Electrophoresis 30. Protein Analysis Using High-Resolution Two-Dimensional Polyacrylamide Gel Electrophoresis and Isoelectric Focusing 31. Protein Gel Staining Methods: An Introduction And Overview 32. Elution of Proteins From Gels 33. Immunodetection By Protein Blotting Performing and Optimizing Western Blots With An Emphasis on Chemiluminescent Detection
Section IX: Purification Procedures: Membrane Proteins And Glycoproteins 34. Detergents: An Overview 35. Purification of Membrane Proteins 36. Purification of Recombinant G-Protein-Coupled Receptors 37. Cell-Free Translation Of Integral Membrane Proteins Into Unilamelar Liposomes
Section X: Characterization of Purified Proteins 38. Determination of Protein Purity 39. Determination of Size, Molecular Weight, and Presence of Subunits 40. Identification and Quantification of Protein Posttranslational Modifications 41. Parallel Methods For Expression And Purification 42. Techniques To Isolate O2-Sensitive Proteins: [4Fe-4S]-FNR As An Example 43. Rethinking Your Purification Procedure 44. Important But Little Known (Or Forgotten) Artifacts in Protein Biochemistry
