Biomolecular and bioanalytical techniques : theory, methodology and applications

Content: List of Contributors xixPreface xxiii1 Principles of Health and Safety and Good Laboratory Practice 1Elaine Armstrong1.1 Introduction 11.2 Good Laboratory Practice 11.3 Risk Assessment 21.4 Chemical Risk Assessment 51.5 Biological Materials and Genetically Modified Organisms 71.6 Vacuum Apparatus, Pressure Systems and Associated Glassware 91.7 Cryogenic Liquefied Gases 91.8 Compressed Gas Cylinders 101.9 Electromagnetic Radiation 101.10 Lasers 111.11 High Magnetic Fields 111.12 Sharps 121.13 Ergonomic Issues 12References 152 Applications of Chemoinformatics in Drug Discovery 17Valerie J. Gillet2.1 Significance and Background 172.2 Computer Representation of Chemical Structures 172.3 Database Searching 182.4 Practical Issues on Representation 222.5 Virtual Screening 222.6 Ligand-Based Virtual Screening 232.7 Protein-Ligand Docking 302.8 Evaluating Virtual Screening Methods 322.9 Case Studies of Virtual Screening 332.10 Conclusions 34References 34Further Reading 363 Bioinformatics and Its Applications in Genomics 37David J. Parry-Smith3.1 Significance and Short Background 373.2 Theory/Principles 403.3 Databases 433.4 Techniques 453.5 Applications 533.6 Concluding Remarks 56References 56Further Reading 57Websites 574 Gene Cloning for the Analysis of Gene Expression 59Huw B. Thomas and Raymond T. O\'Keefe4.1 Identifying Target Sequence 594.2 In Silico Design 594.3 Primer Design 604.4 Template Preparation 624.5 Cloning Methods 644.6 Uses for Cloned DNA Sequences 664.7 Verifying Cloned Sequences 674.8 Applications of Gene Constructs 684.9 Case Study: Cloning of a Human Missense Variant Exon into a Minigene Splicing Vector 694.10 Case Study: Epitope Tagging of a Yeast Gene 74References 79Further Reading 79Websites 805 Proteomic Techniques and Their Applications 81Hsueh-Fen Juan5.1 Significance and Background 815.2 Principles of Major Proteomics Techniques 825.3 Methods for Proteomics 855.4 Applications 925.5 Concluding Remarks 95Acknowledgements 95References 95Further Reading 99Website Resources 996 Overproduction, Separation and Purification of Affinity-Tagged Proteins from Escherichia coli 101Finbarr Hayes and Daniela Barilla6.1 Introduction 1016.2 Selecting an Affinity Tag: Glutathione-S-Transferase, Maltose-Binding Protein and Hexa-Histidine Motifs 1036.3 The pET Vector Series: Archetypal Expression Vectors in E. coli 1056.4 IMAC of a His6-Tagged Protein: Example Methodology with the ParF DNA Segregation Protein 1076.5 Production and Purification of a GST-Tagged Protein: Example Methodology with the C-Terminal Domain of Yeast RNA Polymerase II 1126.6 Further Purification of Tagged Proteins 1146.7 Alternative Hosts for Protein Production 1166.8 Concluding Remarks 116Acknowledgements 117References 117Further Reading 1217 Chromatography: Separation Techniques in Biology 123W John Lough and Mark Carlile7.1 Introduction to Chromatographic Separation 1237.2 General Considerations for Protein Separation by Chromatography 1327.3 Engineering Proteins for Streamlined Chromatographic Separations 1427.4 Example Chromatographic Separations of Biological Samples 1437.5 Other Applications of Chromatography for Biological Sample Preparation and Analysis 147References 147Further Reading 1528 Synthetic Methodology in Chemical Biology 153Richard C. Brewster and Stephen Wallace8.1 Introduction 1538.2 Peptide Synthesis 1538.3 Amide Bond Synthesis 1548.4 Bioorthogonal Chemistry 1648.5 The Copper-Catalysed Azide-Alkyne Cycloaddition Reaction (CuAAC) 1658.6 Unnatural Amino Acid Incorporation 1698.7 Case Studies 1728.8 Conclusion 176References 177Further Reading 1789 Reaction Chemical Kinetics in Biology 179Nicholas J. Harmer and Mirella Vivoli Vega9.1 Significance 1799.2 Overview of Kinetics and Its Application to Biology 1809.3 Determination of Enzyme Kinetic Mechanisms 1889.4 Technique/Protocol: Determination of Michaelis-Menten Parameters for a Bisubstrate Enzyme and Use of Product Inhibition to Determine Mechanism 2019.5 Case Study: Determination of Michaelis-Menten Parameters for a Bisubstrate Enzyme 2109.6 More Advanced Methods 2129.7 Concluding Remarks 214References 21410 Mass Spectrometry and Its Applications 219Blagojce Jovcevski and Tara L. Pukala10.1 Significance 21910.2 Theories and Principles of Biomolecular Mass Spectrometry 22110.3 Techniques and Methodology in Biomolecular Mass Spectrometry 23410.4 Applications 24210.5 Concluding Remarks 248Abbreviations 248References 249Further Reading 25311 Applications and Complementarity of Analytical Ultracentrifugation and Light-Scattering Techniques 255Chad A. Brautigam11.1 Introduction 25511.2 Analytical Ultracentrifugation 25511.3 Light Scattering 26211.4 Protocols 26611.5 Applications 27011.6 Conclusions 275Acknowledgements 275References 276Further Reading 27812 Application of Isothermal Titration Calorimetry (ITC) to Biomolecular Interactions 279Graeme L. Conn12.1 Introduction 27912.2 Principles and Theory of ITC 28112.3 Protocols for Design, Implementation and Analysis of ITC Experiments 28512.4 Example Applications of ITC to Analysis of Biomolecular Interactions 29912.5 Concluding Remarks 304Acknowledgements 304References 304Further Reading 305Website Resources 30513 An Introduction to Infra-red and Raman Spectroscopies for Pharmaceutical and Biomedical Studies 307Ka Lung Andrew Chan13.1 Significance and Short Background 30713.2 Theory 30713.3 Technique/Methodology/Protocol 31313.4 Applications 32313.5 Concluding Remarks 327References 327Further Reading 33114 Fluorescence Spectroscopy and Its Applications in Analysing Biomolecular Processes 333Nathan N. Alder14.1 Significance and Background 33314.2 Theory and Principles 33414.3 Techniques, Methodologies and Protocols 34114.4 Case Studies: Fluorescence Spectroscopy to Analyse Membrane Protein Structural Dynamics 35814.5 Concluding Remarks 360Acknowledgements 361References 361Further Reading 36315 Circular Dichroism and Related Spectroscopic Techniques 365Sophia C. Goodchild, Krishanthi Jayasundera and Alison Rodger15.1 Significance and Background 36515.2 Theory/Principles 36615.3 Technique/Methodology/Protocol 37115.4 Applications 37615.5 Concluding Remarks 382References 382Further Reading 38416 Principles and Practice in Macromolecular X-Ray Crystallography 385Arnaud Basle and Richard J. Lewis16.1 Significance and Short Background 38516.2 Theory and Principles: Overview 38516.3 Methodology 39716.4 Applications 41216.5 Concluding Remarks 414Acknowledgements 414References 414Further Reading 41817 Biomolecular NMR Spectroscopy and Structure Determination of DNA 421Tony Cheung and Vasudevan Ramesh17.1 Significance and Background 42117.2 Basic NMR Theory 42217.3 Multidimensional NMR Spectroscopy 42817.4 NMR Instrumentation and Experiments 42917.5 Structure and Conformational Parameters of DNA 43517.6 NMR Structure Determination 44017.7 Case Study 1: NMR Structure Determination and Conformational Analysis of 17mer Canonical GC DNA 44617.8 Case Study 2: NMR Structure Determination and Conformational Analysis of 13mer 6-ThioguanineModified GC DNA 46017.9 Conclusion 464References 466Further Reading 46918 Cryo-TEM and Biological Structure Determination 471SzymonW. Manka and Carolyn A. Moores18.1 Significance and Background 47118.2 Theoretical Principles of Biological Cryo-TEM 47318.3 Experimental Approaches in Biological Cryo-TEM 48118.4 Cryo-TEM Case Studies 49118.5 Concluding Remarks 496Acknowledgements 496References 496Website Resources 49919 Computer Modelling and Molecular Dynamics Simulation of Biomolecules 501Maria Reif and Martin Zacharias19.1 Significance 50119.2 Theory and Principles 50219.3 Methodology 50619.4 Applications 52519.5 Concluding Remarks 528References 529Further Reading 535Index 537