3rd edition. Addison Wesley Longman Inc. 2000. - 475 p.
The undergraduate biochemistry teaching laboratory has become an essential feature in the training of students majoring in biochemistry, chemistry, molecular biology, and related biological sciences.
In the development of an experiment, primary consideration was given to the use of mode procedures and techniques. Therefore, students will lea procedures that are now used in actual laboratory settings, whether academic or industrial. Each experiment presents a challenging laboratory situation or problem to be solved by the students. In each experiment, a technique is introduced that allows students to obtain and evaluate properties of a biomolecule, a biochemical reaction, or a biological process.
Contents
Part One Theory and Experimental Techniques
Introduction to the Biochemistry Laboratory
Safety in the Laboratory
The Laboratory Notebook and Experiment Reports
Details of Experimental Write-up
Cleaning Laboratory Glassware
Glassware
Quartz and Glass Cuvettes
Preparation and Storage of Solutions
Water Quality
Solution Preparation
EQuantitative Transfer of Liquids
Filling a Pipet
Disposable Pasteur Pipets
Calibrated Pipets
Automatic Pipetting Systems
Cleaning and Drying Pipets
Statistical Analysis of Experimental Data
Analysis of Experimental Data
Determination of the Mean, Sample Deviation, and Standard Deviation Statistical Analysis in Practice
Study Problems
Further Reading
On the Web
General Laboratory Procedures
pH, Buffers, Electrodes, and Biosensors
Measurement of pH
Biochemical Buffers Selection of a Biochemical Buffer
The Oxygen Electrode
Ion-Selective Electrodes and Biosensors
Measurement of Protein Solutions
The Biuret and Lowry Assays
The Bradford Assay
The BCA Assay
The Spectrophotometnc Assay
Measurement of Nucleic Acid Solutions
The Spectrophotometnc Assay
Other Assays for Nucleic Acids
Techniques for Sample Peparation
Dialysis
Ultrafiltration
Lyophilization and Centrifugal Vacuum Concentration
Study Problems
Further Reading
Sample Preparation
Purification and Identification of Biomolecules by Chromatography
Introduction to Chromatography
Partition versus Adsorption Chromatography
Planar Chromatography (Paper and Thin-Layer)
Preparation of the Stationary Support
Solvent Development of the Support
Detection and Measurement of Components
Applications of Planar Chromatography
Gas Chromatography (GC)
Instrumentation
Selection of Operating Conditions
Analysis of GC Data
Advantages and Limitations of GC
Column Chromatography
Operation of a Chromatographic Column
Packing the Column
Loading the Column
Eluting the Column
Collecting the Eluent
Detection of Eluting Components
Ion-Exchange Chromatography
Ion-Exchange Resins
Selection of the Ion Exchanger
Choice of Buffer
Preparation of the Ion Exchanger
Using the Ion-Exchange Resin
Storage of Resins
Gel Exclusion Chromatography
Theory of Gel Filtration
Physical Characterization of Gel Chromatography
Chemical Properties of Gels
Selecting a Gel
Gel Preparation and Storage
Operation of a Gel Column
Applications of Gel Exclusion Chromatography
High-Performance Liquid Chromatography (HPLC)
Instrumentation
Stationary Phases in HPLC
The Mobile Phase
Solvents for HPLC Operation
Gradient Elution in HPLC
Sample Preparation and Selection of HPLC Operating Conditions FPLC – A Modification of HPLC
Affinity Chromatography and Immunoadsorption
Chromatographic Media
The Immobilized Ligand
Attachment of Ligand to Matrix
Experimental Procedure for Affinity Chromatography
Perfusion Chromatography
Study Problems
Further Reading
Chromatography on the Web
Characterization of Proteins and Nucleic Acids by Electrophoresis
Theory of Electrophoresis
Methods of Electrophoresis
Polyacrylamide Gel Electrophoresis (PAGE)
Nucleic Acid Sequencing Gels
Agarose Gel Electrophoresis
Pulsed Field Gel Electrophoresis (PFGE)
Isoelectric Focusing of Proteins
Two-Dimensional Electrophoresis of Proteins
Capillary Electrophoresis (CE)
Immunoelectrophoresis (IE)
Practical Aspects of Electrophoresis
Staining and Detecting Electrophoresis Bands
Protein and Nucleic Acid Blotting
Analysis of Electrophoresis Results
Study Problems
Further Reading
Electrophoresis on the Web
Spectroscopic Analysis of Biomolecules
Ultraviolet-Visible Absorption Spectrophotometry Principles
Applications
Fluorescence Spectrophotometry Principles
Instrumentation
Applications
Difficulties in Fluorescence Measurements
Other Spectroscopic Methods
Nuclear Magnetic Resonance Spectroscopy
Mass Spectrometry
Study Problems
Further Reading
Spectroscopy on the Web
Radioisotopes in Biochemical Research
Origin and Properties of Radioactivity
Introduction
Isotopes in Biochemistry
Units of Radioactivity
Detection and Measurement of Radioactivity Liquid Scintillation Counting
Geiger-Muller Counting of Radioactivity
Scintillation Counting of у Rays
Background Radiation
Applications of Radioisotopes
Statistical Analysis of Radioactivity Measurements
Radioisotopes and Safety
Preparation for the Experiment
Performing the Experiment
Study Problems
Further Reading
Radioisotopes on the Web
Centrifugation in Biochemical Research
A Basic Principles of Centrifugation
Instrumentation for Centrifugation
Low-Speed Centrifuges
High-Speed Centrifuges
Ultracentrifuges
Applications of Centrifugation
Preparative Techniques
Analytical Measurements
Care of Centrifuges and Rotors
Study Problems
Further Reading
Centrifugation on the Web
Part Two Experiments
experiment 1 Using the Computer in Biochemical Research
Experiment 2 Structural Analysis of a Dipeptide
Experiment 3 Using Gel Filtration to Study Ligand-Protein Interactions
Experiment 4 Isolation and Characterization of Bovine Milk ?-Lactalbumin
Experiment 5 Kinetic Analysis of Tyrosinase
Experiment 6 Purification and Characterization of Triacylglycerols in Natural Oils
experiment 7 Identification of Serum Glycoproteins by SDS-PAGE and Weste Blotting
experiment 8 Isolation and Characterization of Plant Pigments
Experiment 9 Photomduced Proton Transport through Chloroplast Membranes
Experiment 10 Isolation, Subfractionation, and Enzymatic Analysis of Beef Heart Mitochondria
Experiment 11 Measurement of Cholesterol and Vitamin С in Biological Samples
Experiment 12 Activity and Thermal Stability of Gel-immobilized Peroxidase
Experiment 13 Extraction and Characterization of Bacterial DNA
Experiment 14 Plasmid DNA Isolation and Characterization by Electrophoresis
Experiment 15 The Action of Restriction Endonucleases on Plasmid or Viral DNA
Appendices
Appendix I Properties of Common Acids and Bases
Appendix II Properties of Common Buffer Compounds
Appendix III pKa Values and pH, Values of Amino Acids
Appendix IV Molecular Weights of Some Common Proteins
Appendix V Common Abbreviations Used in This Text
Appendix VI Units of Measurement
Appendix VII Table of the Elements
Appendix VIII Values of t-for Analysis of Statistical Confidence Limits
Appendix IX Answers to Selected Study Problems
Index
The undergraduate biochemistry teaching laboratory has become an essential feature in the training of students majoring in biochemistry, chemistry, molecular biology, and related biological sciences.
In the development of an experiment, primary consideration was given to the use of mode procedures and techniques. Therefore, students will lea procedures that are now used in actual laboratory settings, whether academic or industrial. Each experiment presents a challenging laboratory situation or problem to be solved by the students. In each experiment, a technique is introduced that allows students to obtain and evaluate properties of a biomolecule, a biochemical reaction, or a biological process.
Contents
Part One Theory and Experimental Techniques
Introduction to the Biochemistry Laboratory
Safety in the Laboratory
The Laboratory Notebook and Experiment Reports
Details of Experimental Write-up
Cleaning Laboratory Glassware
Glassware
Quartz and Glass Cuvettes
Preparation and Storage of Solutions
Water Quality
Solution Preparation
EQuantitative Transfer of Liquids
Filling a Pipet
Disposable Pasteur Pipets
Calibrated Pipets
Automatic Pipetting Systems
Cleaning and Drying Pipets
Statistical Analysis of Experimental Data
Analysis of Experimental Data
Determination of the Mean, Sample Deviation, and Standard Deviation Statistical Analysis in Practice
Study Problems
Further Reading
On the Web
General Laboratory Procedures
pH, Buffers, Electrodes, and Biosensors
Measurement of pH
Biochemical Buffers Selection of a Biochemical Buffer
The Oxygen Electrode
Ion-Selective Electrodes and Biosensors
Measurement of Protein Solutions
The Biuret and Lowry Assays
The Bradford Assay
The BCA Assay
The Spectrophotometnc Assay
Measurement of Nucleic Acid Solutions
The Spectrophotometnc Assay
Other Assays for Nucleic Acids
Techniques for Sample Peparation
Dialysis
Ultrafiltration
Lyophilization and Centrifugal Vacuum Concentration
Study Problems
Further Reading
Sample Preparation
Purification and Identification of Biomolecules by Chromatography
Introduction to Chromatography
Partition versus Adsorption Chromatography
Planar Chromatography (Paper and Thin-Layer)
Preparation of the Stationary Support
Solvent Development of the Support
Detection and Measurement of Components
Applications of Planar Chromatography
Gas Chromatography (GC)
Instrumentation
Selection of Operating Conditions
Analysis of GC Data
Advantages and Limitations of GC
Column Chromatography
Operation of a Chromatographic Column
Packing the Column
Loading the Column
Eluting the Column
Collecting the Eluent
Detection of Eluting Components
Ion-Exchange Chromatography
Ion-Exchange Resins
Selection of the Ion Exchanger
Choice of Buffer
Preparation of the Ion Exchanger
Using the Ion-Exchange Resin
Storage of Resins
Gel Exclusion Chromatography
Theory of Gel Filtration
Physical Characterization of Gel Chromatography
Chemical Properties of Gels
Selecting a Gel
Gel Preparation and Storage
Operation of a Gel Column
Applications of Gel Exclusion Chromatography
High-Performance Liquid Chromatography (HPLC)
Instrumentation
Stationary Phases in HPLC
The Mobile Phase
Solvents for HPLC Operation
Gradient Elution in HPLC
Sample Preparation and Selection of HPLC Operating Conditions FPLC – A Modification of HPLC
Affinity Chromatography and Immunoadsorption
Chromatographic Media
The Immobilized Ligand
Attachment of Ligand to Matrix
Experimental Procedure for Affinity Chromatography
Perfusion Chromatography
Study Problems
Further Reading
Chromatography on the Web
Characterization of Proteins and Nucleic Acids by Electrophoresis
Theory of Electrophoresis
Methods of Electrophoresis
Polyacrylamide Gel Electrophoresis (PAGE)
Nucleic Acid Sequencing Gels
Agarose Gel Electrophoresis
Pulsed Field Gel Electrophoresis (PFGE)
Isoelectric Focusing of Proteins
Two-Dimensional Electrophoresis of Proteins
Capillary Electrophoresis (CE)
Immunoelectrophoresis (IE)
Practical Aspects of Electrophoresis
Staining and Detecting Electrophoresis Bands
Protein and Nucleic Acid Blotting
Analysis of Electrophoresis Results
Study Problems
Further Reading
Electrophoresis on the Web
Spectroscopic Analysis of Biomolecules
Ultraviolet-Visible Absorption Spectrophotometry Principles
Applications
Fluorescence Spectrophotometry Principles
Instrumentation
Applications
Difficulties in Fluorescence Measurements
Other Spectroscopic Methods
Nuclear Magnetic Resonance Spectroscopy
Mass Spectrometry
Study Problems
Further Reading
Spectroscopy on the Web
Radioisotopes in Biochemical Research
Origin and Properties of Radioactivity
Introduction
Isotopes in Biochemistry
Units of Radioactivity
Detection and Measurement of Radioactivity Liquid Scintillation Counting
Geiger-Muller Counting of Radioactivity
Scintillation Counting of у Rays
Background Radiation
Applications of Radioisotopes
Statistical Analysis of Radioactivity Measurements
Radioisotopes and Safety
Preparation for the Experiment
Performing the Experiment
Study Problems
Further Reading
Radioisotopes on the Web
Centrifugation in Biochemical Research
A Basic Principles of Centrifugation
Instrumentation for Centrifugation
Low-Speed Centrifuges
High-Speed Centrifuges
Ultracentrifuges
Applications of Centrifugation
Preparative Techniques
Analytical Measurements
Care of Centrifuges and Rotors
Study Problems
Further Reading
Centrifugation on the Web
Part Two Experiments
experiment 1 Using the Computer in Biochemical Research
Experiment 2 Structural Analysis of a Dipeptide
Experiment 3 Using Gel Filtration to Study Ligand-Protein Interactions
Experiment 4 Isolation and Characterization of Bovine Milk ?-Lactalbumin
Experiment 5 Kinetic Analysis of Tyrosinase
Experiment 6 Purification and Characterization of Triacylglycerols in Natural Oils
experiment 7 Identification of Serum Glycoproteins by SDS-PAGE and Weste Blotting
experiment 8 Isolation and Characterization of Plant Pigments
Experiment 9 Photomduced Proton Transport through Chloroplast Membranes
Experiment 10 Isolation, Subfractionation, and Enzymatic Analysis of Beef Heart Mitochondria
Experiment 11 Measurement of Cholesterol and Vitamin С in Biological Samples
Experiment 12 Activity and Thermal Stability of Gel-immobilized Peroxidase
Experiment 13 Extraction and Characterization of Bacterial DNA
Experiment 14 Plasmid DNA Isolation and Characterization by Electrophoresis
Experiment 15 The Action of Restriction Endonucleases on Plasmid or Viral DNA
Appendices
Appendix I Properties of Common Acids and Bases
Appendix II Properties of Common Buffer Compounds
Appendix III pKa Values and pH, Values of Amino Acids
Appendix IV Molecular Weights of Some Common Proteins
Appendix V Common Abbreviations Used in This Text
Appendix VI Units of Measurement
Appendix VII Table of the Elements
Appendix VIII Values of t-for Analysis of Statistical Confidence Limits
Appendix IX Answers to Selected Study Problems
Index