Biomolecular and Bioanalytical Techniques Theory, Methodology and Applications

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. 

Editor:

Dr Vasudevan Ramesh, retired from the Faculty of the School of Chemistry at the University of Manchester, Manchester, UK, after 18 years of distinguished service. He taught Physical Chemistry at all degree levels and pursued an active research programme in Biomolecular NMR spectroscopy. He was a Leverhulme Research Fellow at the University of Leicester and a Visiting Lecturer and Annual Award winner at the Peking University, Beijing, China.

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

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