An essential guide to biomolecular and bioanalytical techniques and their applications
Biomolecular and Bioanalytical Techniques offers an introduction to, and a basic understanding of, a wide range of biophysical techniques. The text takes an interdisciplinary approach with contributions from a panel of distinguished experts. With a focus on research, the text comprehensively covers a broad selection of topics drawn from contemporary research in the fields of chemistry and biology. Each of the internationally reputed authors has contributed a single chapter on a specific technique. The chapters cover the specific technique’s background, theory, principles, technique, methodology, protocol and applications.
The text explores the use of a variety of analytical tools to characterise biological samples. The contributors explain how to identify and quantify biochemically important molecules, including small molecules as well as biological macromolecules such as enzymes, antibodies, proteins, peptides and nucleic acids. This book is filled with essential knowledge and explores the skills needed to carry out the research and development roles in academic and industrial laboratories.
- A technique-focused book that bridges the gap between an introductory text and a book on advanced research methods
- Provides the necessary background and skills needed to advance the research methods
- Features a structured approach within each chapter
- Demonstrates an interdisciplinary approach that serves to develop independent thinking
Written for students in chemistry, biological, medical, pharmaceutical, forensic and biophysical sciences, Biomolecular and Bioanalytical Techniques is an in-depth review of the most current biomolecular and bioanalytical techniques in the field.
Table of Contents
List of Contributors xix
Preface xxiii
1 Principles of Health and Safety and Good Laboratory Practice 1
Elaine Armstrong
1.1 Introduction 1
1.2 Good Laboratory Practice 1
1.3 Risk Assessment 2
1.4 Chemical Risk Assessment 5
1.5 Biological Materials and Genetically Modified Organisms 7
1.6 Vacuum Apparatus, Pressure Systems and Associated Glassware 9
1.7 Cryogenic Liquefied Gases 9
1.8 Compressed Gas Cylinders 10
1.9 Electromagnetic Radiation 10
1.10 Lasers 11
1.11 High Magnetic Fields 11
1.12 Sharps 12
1.13 Ergonomic Issues 12
References 15
2 Applications of Chemoinformatics in Drug Discovery 17
Valerie J. Gillet
2.1 Significance and Background 17
2.2 Computer Representation of Chemical Structures 17
2.3 Database Searching 18
2.4 Practical Issues on Representation 22
2.5 Virtual Screening 22
2.6 Ligand-Based Virtual Screening 23
2.7 Protein-Ligand Docking 30
2.8 Evaluating Virtual Screening Methods 32
2.9 Case Studies of Virtual Screening 33
2.10 Conclusions 34
References 34
Further Reading 36
3 Bioinformatics and Its Applications in Genomics 37
David J. Parry-Smith
3.1 Significance and Short Background 37
3.2 Theory/Principles 40
3.3 Databases 43
3.4 Techniques 45
3.5 Applications 53
3.6 Concluding Remarks 56
References 56
Further Reading 57
Websites 57
4 Gene Cloning for the Analysis of Gene Expression 59
Huw B. Thomas and Raymond T. O’Keefe
4.1 Identifying Target Sequence 59
4.2 In Silico Design 59
4.3 Primer Design 60
4.4 Template Preparation 62
4.5 Cloning Methods 64
4.6 Uses for Cloned DNA Sequences 66
4.7 Verifying Cloned Sequences 67
4.8 Applications of Gene Constructs 68
4.9 Case Study: Cloning of a Human Missense Variant Exon into a Minigene Splicing Vector 69
4.10 Case Study: Epitope Tagging of a Yeast Gene 74
References 79
Further Reading 79
Websites 80
5 Proteomic Techniques and Their Applications 81
Hsueh-Fen Juan
5.1 Significance and Background 81
5.2 Principles of Major Proteomics Techniques 82
5.3 Methods for Proteomics 85
5.4 Applications 92
5.5 Concluding Remarks 95
Acknowledgements 95
References 95
Further Reading 99
Website Resources 99
6 Overproduction, Separation and Purification of Affinity-Tagged Proteins from Escherichia coli 101
Finbarr Hayes and Daniela Barillà
6.1 Introduction 101
6.2 Selecting an Affinity Tag: Glutathione-S-Transferase, Maltose-Binding Protein and Hexa-Histidine Motifs 103
6.3 The pET Vector Series: Archetypal Expression Vectors in E. coli 105
6.4 IMAC of a His6-Tagged Protein: Example Methodology with the ParF DNA Segregation Protein 107
6.5 Production and Purification of a GST-Tagged Protein: Example Methodology with the C-Terminal Domain of Yeast RNA Polymerase II 112
6.6 Further Purification of Tagged Proteins 114
6.7 Alternative Hosts for Protein Production 116
6.8 Concluding Remarks 116
Acknowledgements 117
References 117
Further Reading 121
7 Chromatography: Separation Techniques in Biology 123
W John Lough and Mark Carlile
7.1 Introduction to Chromatographic Separation 123
7.2 General Considerations for Protein Separation by Chromatography 132
7.3 Engineering Proteins for Streamlined Chromatographic Separations 142
7.4 Example Chromatographic Separations of Biological Samples 143
7.5 Other Applications of Chromatography for Biological Sample Preparation and Analysis 147
References 147
Further Reading 152
8 Synthetic Methodology in Chemical Biology 153
Richard C. Brewster and Stephen Wallace
8.1 Introduction 153
8.2 Peptide Synthesis 153
8.3 Amide Bond Synthesis 154
8.4 Bioorthogonal Chemistry 164
8.5 The Copper-Catalysed Azide-Alkyne Cycloaddition Reaction (CuAAC) 165
8.6 Unnatural Amino Acid Incorporation 169
8.7 Case Studies 172
8.8 Conclusion 176
References 177
Further Reading 178
9 Reaction Chemical Kinetics in Biology 179
Nicholas J. Harmer and Mirella Vivoli Vega
9.1 Significance 179
9.2 Overview of Kinetics and Its Application to Biology 180
9.3 Determination of Enzyme Kinetic Mechanisms 188
9.4 Technique/Protocol: Determination of Michaelis-Menten Parameters for a Bisubstrate Enzyme and Use of Product Inhibition to Determine Mechanism 201
9.5 Case Study: Determination of Michaelis-Menten Parameters for a Bisubstrate Enzyme 210
9.6 More Advanced Methods 212
9.7 Concluding Remarks 214
References 214
10 Mass Spectrometry and Its Applications 219
Blagojce Jovcevski and Tara L. Pukala
10.1 Significance 219
10.2 Theories and Principles of Biomolecular Mass Spectrometry 221
10.3 Techniques and Methodology in Biomolecular Mass Spectrometry 234
10.4 Applications 242
10.5 Concluding Remarks 248
Abbreviations 248
References 249
Further Reading 253
11 Applications and Complementarity of Analytical Ultracentrifugation and Light-Scattering Techniques 255
Chad A. Brautigam
11.1 Introduction 255
11.2 Analytical Ultracentrifugation 255
11.3 Light Scattering 262
11.4 Protocols 266
11.5 Applications 270
11.6 Conclusions 275
Acknowledgements 275
References 276
Further Reading 278
12 Application of Isothermal Titration Calorimetry (ITC) to Biomolecular Interactions 279
Graeme L. Conn
12.1 Introduction 279
12.2 Principles and Theory of ITC 281
12.3 Protocols for Design, Implementation and Analysis of ITC Experiments 285
12.4 Example Applications of ITC to Analysis of Biomolecular Interactions 299
12.5 Concluding Remarks 304
Acknowledgements 304
References 304
Further Reading 305
Website Resources 305
13 An Introduction to Infra-red and Raman Spectroscopies for Pharmaceutical and Biomedical Studies 307
Ka Lung Andrew Chan
13.1 Significance and Short Background 307
13.2 Theory 307
13.3 Technique/Methodology/Protocol 313
13.4 Applications 323
13.5 Concluding Remarks 327
References 327
Further Reading 331
14 Fluorescence Spectroscopy and Its Applications in Analysing Biomolecular Processes 333
Nathan N. Alder
14.1 Significance and Background 333
14.2 Theory and Principles 334
14.3 Techniques, Methodologies and Protocols 341
14.4 Case Studies: Fluorescence Spectroscopy to Analyse Membrane Protein Structural Dynamics 358
14.5 Concluding Remarks 360
Acknowledgements 361
References 361
Further Reading 363
15 Circular Dichroism and Related Spectroscopic Techniques 365
Sophia C. Goodchild, Krishanthi Jayasundera and Alison Rodger
15.1 Significance and Background 365
15.2 Theory/Principles 366
15.3 Technique/Methodology/Protocol 371
15.4 Applications 376
15.5 Concluding Remarks 382
References 382
Further Reading 384
16 Principles and Practice in Macromolecular X-Ray Crystallography 385
Arnaud Baslé and Richard J. Lewis
16.1 Significance and Short Background 385
16.2 Theory and Principles: Overview 385
16.3 Methodology 397
16.4 Applications 412
16.5 Concluding Remarks 414
Acknowledgements 414
References 414
Further Reading 418
17 Biomolecular NMR Spectroscopy and Structure Determination of DNA 421
Tony Cheung and Vasudevan Ramesh
17.1 Significance and Background 421
17.2 Basic NMR Theory 422
17.3 Multidimensional NMR Spectroscopy 428
17.4 NMR Instrumentation and Experiments 429
17.5 Structure and Conformational Parameters of DNA 435
17.6 NMR Structure Determination 440
17.7 Case Study 1: NMR Structure Determination and Conformational Analysis of 17mer Canonical GC DNA 446
17.8 Case Study 2: NMR Structure Determination and Conformational Analysis of 13mer 6-ThioguanineModified GC DNA 460
17.9 Conclusion 464
References 466
Further Reading 469
18 Cryo-TEM and Biological Structure Determination 471
SzymonW. Manka and Carolyn A. Moores
18.1 Significance and Background 471
18.2 Theoretical Principles of Biological Cryo-TEM 473
18.3 Experimental Approaches in Biological Cryo-TEM 481
18.4 Cryo-TEM Case Studies 491
18.5 Concluding Remarks 496
Acknowledgements 496
References 496
Website Resources 499
19 Computer Modelling and Molecular Dynamics Simulation of Biomolecules 501
Maria Reif and Martin Zacharias
19.1 Significance 501
19.2 Theory and Principles 502
19.3 Methodology 506
19.4 Applications 525
19.5 Concluding Remarks 528
References 529
Further Reading 535
Index 537
