Basic Bioscience Laboratory Techniques (eBook)

Philip L. R. Bonner, PhD, is a member of the Interdisciplinary Biomedical Sciences Centre, Protein Purification and Proteomics Research Group. Alan J. Hargreaves, PhD, is Associate Professor in Biochemistry, School of Science & Technology, Nottingham Trent University in the United Kingdom. He is also Chair of the College Research Ethics Committee.

COVER 1
TITLE PAGE 5
COPYRIGHT PAGE 6
CONTENTS 7
PREFACE 9
GLOSSARY 11
ABBREVIATIONS 15
ABOUT THE COMPANION WEBSITE 19
CHAPTER 1 THE PREPARATION OF SOLUTIONS IN BIOSCIENCE RESEARCH 21
1.1 Introduction 21
1.2 Concentration 22
1.2.1 Molarity 22
1.3 Using a Balance to Weigh Out Reagents 22
1.3.1 The Use of an Electronic Balance 22
1.3.2 The Use of a ‘Top Pan’ Balance 24
1.3.3 The Use of Analytical Balances 24
1.4 Practical Considerations When Making a 1.0 M Solution 25
1.4.1 Preparing Solutions with a Concentration Less than 1.0 M and a Volume Less than a Litre 26
1.4.2 Preparing Molar Solutions of Liquids 28
1.4.3 Preparing Percentage Solutions 28
1.5 Dilutions and the Use of Pipettes 29
1.5.1 The Use of Graduated Cylindrical Glass or Plastic Pipettes 31
1.5.2 The Use of Handheld Micropipettes 31
1.5.3 Diluting Down from a Stock Solution 32
1.6 Water, Acids and Bases 33
1.6.1 Water as a Solvent 33
1.6.2 The Ionization of Water 33
1.6.3 Acids and Bases 35
1.7 Buffers 36
1.7.1 Preparing a Buffer and Using a pH Meter 37
1.8 The Equilibrium/Dissociation Constant (Ka) for an Acid or Base and the Henderson–Hasselbalch Equation 38
1.9 Summary 40
Notes 40
CHAPTER 2 MICROSCOPY 41
2.1 Introduction 41
2.2 Microscopes – General Principles 42
2.3 Principles of Image Formation 43
2.3.1 Units of Measurement 44
2.4 Light Microscopy 45
2.4.1 Bright Field Microscopy 45
2.4.2 Phase Contrast Microscopy 48
2.4.3 Differential Interference Contrast 48
2.4.4 Fluorescence Microscopy 49
2.4.5 How to Use a Light Microscope 50
2.4.6 Data Analysis and Presentation 53
2.5 Electron Microscopy 56
2.6 Summary 59
CHAPTER 3 SPECTROPHOTOMETRY 61
3.1 Introduction 61
3.2 The Electromagnetic Spectrum 61
3.3 The Absorbance of Light 63
3.3.1 Fluorescence 63
3.3.2 Luminescence 65
3.4 Absorption Spectrophotometry 65
3.5 The Laws Governing the Absorbance of Light 65
3.5.1 Attenuance of Light (Optical Density) 66
3.6 The Beer–Lambert Law 67
3.6.1 Limitations of the Beer–Lambert Law 68
3.7 Spectrophotometers 69
3.7.1 Introduction 69
3.7.2 The Light Source 70
3.7.3 The Optics Used to Generate Wavelengths of Light 70
3.7.4 The Slit Width 70
3.7.5 The Cuvette 70
3.7.6 The Detector 72
3.7.7 The Use of High-throughput Microplate Readers 72
3.8 Applications of Spectrophotometry in Bioscience 74
3.8.1 Direct Measurements of Biological Molecules 74
3.8.2 Chromophore Assays 76
3.8.3 Enzyme Catalyzed Reaction 77
3.9 Summary 82
Notes 82
CHAPTER 4 DATA ANALYSIS AND PRESENTATION 83
4.1 Introduction 83
4.2 Statistical Analysis of Data: Some Key Definitions 84
4.2.1 Populations and Samples 84
4.2.2 Variables and Observations 84
4.2.3 Descriptive Statistics 84
4.2.4 Measures of Spread (Dispersion) 85
4.3 Distributions 87
4.3.1 Normal Distribution 87
4.3.2 Asymmetric (Non-Normal) Distribution 90
4.3.3 Discrete Probability Distributions 90
4.4 Statistical Comparison of Data 90
4.4.1 Comparing Two Sets of Values 91
4.4.2 Unpaired and Paired Non-Parametric Tests 93
4.4.3 Comparisons of Multiple Sets of Data 94
4.4.4 Chi Square Test for Dichotomous Variables 94
4.4.5 Analyzing Relationships Between Two Variables: Linear Correlation and Regression 95
4.5 Presentation, Structure and Organization of Data in Laboratory Reports 97
4.5.1 Data Presentation 98
4.5.2 Content 98
4.5.3 Articles in Journals 101
4.5.4 Articles in Books 101
4.5.5 Reference Management Software 101
4.6 Summary 102
CHAPTER 5 THE EXTRACTION AND CLARIFICATION OF BIOLOGICAL MATERIAL 103
5.1 Introduction 103
5.2 Extraction 103
5.2.1 Proteins 103
5.2.2 Deoxyribonucleic Acid 104
5.2.3 Lipids 104
5.2.4 Organelles 104
5.3 The Extraction Methods Used for Animal and Plant Tissue 104
5.3.1 Mortar and Pestle 105
5.3.2 Homogenizers and Tissue Grinders 105
5.3.3 Blenders 106
5.4 The Extraction Methods for Bacteria 106
Key Points to Remember 5.2Extraction 106
5.5 Clarification 107
5.5.1 Introduction to Centrifugation 107
5.5.2 Centrifuges 109
5.5.3 Centrifuge Rotors 110
5.5.4 Centrifuge Tubes 111
5.6 Centrifugation Techniques 112
5.6.1 Differential Centrifugation 112
5.6.2 Density Gradient Centrifugation 113
5.6.3 Isopycnic Centrifugation (Equilibrium Density Gradient Centrifugation) 113
5.7 Points of Good Practice in Centrifugation 113
5.8 Summary 114
Notes 115
CHAPTER 6 ELECTROPHORESIS OF PROTEINS AND NUCLEIC ACIDS 117
6.1 General Introduction 117
6.2 Separation of Protein Mixtures by Gel Electrophoresis 117
6.2.1 Preparation of Electrophoresis Gels 118
6.2.2 Sample Preparation and Loading 120
6.2.3 Running Electrophoresis Gels 122
6.3 Other Electrophoretic Techniques Applied to Proteins 126
6.3.1 Non-Denaturing Gel Electrophoresis 126
6.3.2 Cellulose Acetate Electrophoresis 129
6.3.3 Western Blotting 129
6.3.4 Isoelectric Focusing (IEF) and Two-Dimensional Polyacrylamide Gel Electrophoresis (2D-PAGE) 129
6.4 Separation of Nucleic Acids by Gel Electrophoresis 130
6.4.1 Sample Preparation 131
6.4.2 Preparation and Running of Agarose Gels 132
6.4.3 Staining of Gels to Detect Nucleic Acids 132
6.5 Applications of Gel Electrophoresis of Nucleic Acids 133
6.5.1 DNA Fingerprinting and PCR 133
6.5.2 Analysis of RNA 134
6.5.3 Pulsed Field Gel Electrophoresis (PFGE) 134
6.5.4 Identification of Specific Sequences Using Hybridization Techniques 134
6.5.5 DNA Sequencing 135
6.6 Summary 135
CHAPTER 7 CHROMATOGRAPHY 137
7.1 Introduction 137
7.2 The Theory of Chromatography 137
7.2.1 The Partition Coefficient (KD) in Chromatography 138
7.3 Factors to Consider in Chromatography 140
7.4 The Methods Used to Elute Samples in Chromatography 142
7.5 A Selection of Methods that Can be Used to Exchange Buffers and Concentrate Samples Prior to Chromatography 143
7.5.1 Dialysis 143
7.5.2 Size Exclusion Chromatography 144
7.5.3 Ultrafiltration 145
7.5.4 Centrifugal Evaporating Machines 145
7.5.5 Lyophilization (Freeze-Drying) 147
7.6 Vacuum Pumps 147
7.7 Different Types of Chromatography and What Properties Can be Used to Separate Molecules 148
7.7.1 Adsorption Chromatography 149
7.7.2 Partition Chromatography 149
7.7.3 Ion Exchange Chromatography 149
7.7.4 Size Exclusion Chromatography (SEC) 149
7.7.5 Affinity Chromatography 150
7.8 Thin-layer Chromatography (TLC) 150
7.9 High-Pressure Liquid Chromatography (HPLC) 151
7.9.1 Normal Phase (NP), Reversed Phase (RP) Hydrophobic Interaction (HIC) and Hydrophilic Interaction Liquid (HILIC) Chromatography 152
7.10 Gas/Liquid Chromatography (GLC) 153
7.11 Ion Exchange Chromatography (IEX) 154
7.11.1 The Purification of Antibodies 156
7.12 Size Exclusion Chromatography (SEC) 156
7.13 Affinity Chromatography 158
7.14 Summary 159
Notes 159
CHAPTER 8 CELL CULTURE TECHNIQUES 161
8.1 Introduction 161
8.2 Growth and Maintenance of Cells in Culture 165
8.2.1 Solidified Media 165
8.2.2 Other Applications of Solid Media Culture Methods 169
8.2.3 Liquid Media 171
8.2.4 Liquid Culture of Eukaryotic Cells 174
8.2.5 Seeding of Cells for Experiments 175
8.2.6 Cryopreservation of Cells 176
8.3 Summary 177
CHAPTER 9 ANTIBODY-BASED ASSAYS (IMMUNOASSAYS) 179
9.1 Antibody Structure and Uses 179
9.2 Antibody Purification, Labelling and Detection 180
9.3 Immunoassay Methods 182
9.3.1 Competitive Assays 183
9.3.2 Non-competitive Immunoassays 187
9.4 Controls 195
9.5 Summary 195
SUGGESTIONS FOR FURTHER READING 197
INDEX 201
EULA 208