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End User License Agreement
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End User License Agreement
by Vasudevan Ramesh
Biomolecular and Bioanalytical Techniques
Cover
List of Contributors
Preface
1 Principles of Health and Safety and Good Laboratory Practice
1.1 Introduction
1.2 Good Laboratory Practice
1.3 Risk Assessment
1.4 Chemical Risk Assessment
1.5 Biological Materials and Genetically Modified Organisms
1.6 Vacuum Apparatus, Pressure Systems and Associated Glassware
1.7 Cryogenic Liquefied Gases
1.8 Compressed Gas Cylinders
1.9 Electromagnetic Radiation
1.10 Lasers
1.11 High Magnetic Fields
1.12 Sharps
1.13 Ergonomic Issues
References
2 Applications of Chemoinformatics in Drug Discovery
2.1 Significance and Background
2.2 Computer Representation of Chemical Structures
2.3 Database Searching
2.4 Practical Issues on Representation
2.5 Virtual Screening
2.6 Ligand‐Based Virtual Screening
2.7 Protein–Ligand Docking
2.8 Evaluating Virtual Screening Methods
2.9 Case Studies of Virtual Screening
2.10 Conclusions
References
Further Reading
3 Bioinformatics and Its Applications in Genomics
3.1 Significance and Short Background
3.2 Theory/Principles
3.3 Databases
3.4 Techniques
3.5 Applications
3.6 Concluding Remarks
References
Further Reading
Websites
4 Gene Cloning for the Analysis of Gene Expression
4.1 Identifying Target Sequence
4.2 In Silico Design
4.3 Primer Design
4.4 Template Preparation
4.5 Cloning Methods
4.6 Uses for Cloned DNA Sequences
4.7 Verifying Cloned Sequences
4.8 Applications of Gene Constructs
4.9 Case Study: Cloning of a Human Missense Variant Exon into a Minigene Splicing Vector
4.10 Case Study: Epitope Tagging of a Yeast Gene
References
Further Reading
Websites
5 Proteomic Techniques and Their Applications
5.1 Significance and Background
5.2 Principles of Major Proteomics Techniques
5.3 Methods for Proteomics
5.4 Applications
5.5 Concluding Remarks
Acknowledgements
References
Further Reading
Website Resources
6 Overproduction, Separation and Purification of Affinity‐Tagged Proteins from Escherichia coli
6.1 Introduction
6.2 Selecting an Affinity Tag: Glutathione‐S‐Transferase, Maltose‐Binding Protein and Hexa‐Histidine Motifs
6.3 The pET Vector Series: Archetypal Expression Vectors in E. coli
6.4 IMAC of a His6‐Tagged Protein: Example Methodology with the ParF DNA Segregation Protein‐Tagged Protein: Example Methodology with the ParF DNA Segregation Protein
6.5 Production and Purification of a GST‐Tagged Protein: Example Methodology with the C‐Terminal Domain of Yeast RNA Polymerase II
6.6 Further Purification of Tagged Proteins
6.7 Alternative Hosts for Protein Production
6.8 Concluding Remarks
Acknowledgements
References
Further Reading
7 Chromatography: Separation Techniques in Biology
7.1 Introduction to Chromatographic Separation
7.2 General Considerations for Protein Separation by Chromatography
7.3 Engineering Proteins for Streamlined Chromatographic Separations
7.4 Example Chromatographic Separations of Biological Samples
7.5 Other Applications of Chromatography for Biological Sample Preparation and Analysis
References
Further Reading
8 Synthetic Methodology in Chemical Biology
8.1 Introduction
8.2 Peptide Synthesis
8.3 Amide Bond Synthesis
8.4 Bioorthogonal Chemistry
8.5 The Copper‐Catalysed Azide‐Alkyne Cycloaddition Reaction (CuAAC)
8.6 Unnatural Amino Acid Incorporation
8.7 Case Studies
8.8 Conclusion
References
Further Reading
9 Reaction Chemical Kinetics in Biology
9.1 Significance
9.2 Overview of Kinetics and Its Application to Biology
9.3 Determination of Enzyme Kinetic Mechanisms
9.4 Technique/Protocol: Determination of Michaelis–Menten Parameters for a Bisubstrate Enzyme and Use of Product Inhibition to Determine Mechanism
9.5 Case Study: Determination of Michaelis–Menten Parameters for a Bisubstrate Enzyme
9.6 More Advanced Methods
9.7 Concluding Remarks
References
10 Mass Spectrometry and Its Applications
10.1 Significance
10.2 Theories and Principles of Biomolecular Mass Spectrometry
10.3 Techniques and Methodology in Biomolecular Mass Spectrometry
10.4 Applications
10.5 Concluding Remarks
References
Further Reading
11 Applications and Complementarity of Analytical Ultracentrifugation and Light‐Scattering Techniques
11.1 Introduction
11.2 Analytical Ultracentrifugation
11.3 Light Scattering
11.4 Protocols
11.5 Applications
11.6 Conclusions
Acknowledgements
References
Further Reading
12 Application of Isothermal Titration Calorimetry (ITC) to Biomolecular Interactions
12.1 Introduction
12.2 Principles and Theory of ITC
12.3 Protocols for Design, Implementation and Analysis of ITC Experiments
12.4 Example Applications of ITC to Analysis of Biomolecular Interactions
12.5 Concluding Remarks
Acknowledgements
References
Further Reading
Website Resources
13 An Introduction to Infra‐red and Raman Spectroscopies for Pharmaceutical and Biomedical Studies
13.1 Significance and Short Background
13.2 Theory
13.3 Technique/Methodology/Protocol
13.4 Applications
13.5 Concluding Remarks
References
Further Reading
14 Fluorescence Spectroscopy and Its Applications in Analysing Biomolecular Processes
14.1 Significance and Background
14.2 Theory and Principles
14.3 Techniques, Methodologies and Protocols
14.4 Case Studies: Fluorescence Spectroscopy to Analyse Membrane Protein Structural Dynamics
14.5 Concluding Remarks
Acknowledgements
References
Further Reading
15 Circular Dichroism and Related Spectroscopic Techniques
15.1 Significance and Background
15.2 Theory/Principles
15.3 Technique/Methodology/Protocol
15.4 Applications
15.5 Concluding Remarks
References
Further Reading
16 Principles and Practice in Macromolecular X‐Ray Crystallography
16.1 Significance and Short Background
16.2 Theory and Principles: Overview
16.3 Methodology
16.4 Applications
16.5 Concluding Remarks
Acknowledgements
References
Further Reading
17 Biomolecular NMR Spectroscopy and Structure Determination of DNA
17.1 Significance and Background
17.2 Basic NMR Theory
17.3 Multidimensional NMR Spectroscopy
17.4 NMR Instrumentation and Experiments [23]
17.5 Structure and Conformational Parameters of DNA
17.6 NMR Structure Determination [50–55]
17.7 Case Study 1: NMR Structure Determination and Conformational Analysis of 17mer Canonical GC DNA
17.8 Case Study 2: NMR Structure Determination and Conformational Analysis of 13mer 6‐Thioguanine Modified GC DNA [70–74]
17.9 Conclusion
References
Further Reading
18 Cryo‐TEM and Biological Structure Determination
18.1 Significance and Background
18.2 Theoretical Principles of Biological Cryo‐TEM
18.3 Experimental Approaches in Biological Cryo‐TEM
18.4 Cryo‐TEM Case Studies
18.5 Concluding Remarks
Acknowledgements
References
Website Resources
19 Computer Modelling and Molecular Dynamics Simulation of Biomolecules
19.1 Significance
19.2 Theory and Principles
19.3 Methodology
19.4 Applications
19.5 Concluding Remarks
References
Further Reading
Index
End User License Agreement
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WILEY END USER LICENSE AGREEMENT
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