Practical Biochemistry: Principles and Techniques

Included Index

General principles of biochemical investigations. Nature of biochemistry. Bioenergetics. Methods for investigating metabolism. Practical considerations. In vivo models. In vitro models. Microscopy. Molecular biology and basic techniques. Components and primary structure of nucleic acids. Genes and genome complexity. Nature of the genetic code. Cellular location of nucleic acids. Cellular functions of DNA. Manipulation of nucleic acids: basic tools and techniques. Isolation and separation of nucleic acids. Restriction mapping of DNA fragments. Nucleic acid blotting methods. Gene probe derivation. Labelling DNA gene probe molecules. The polymerase chain reaction. Nucleotide sequencing of DNA. Bioinformatics and the Internet. Molecular cloning and gene analysis. Constructing gene libraries. Cloning vectors. Hybridisation and gene probes. Screening gene libraries. Applications of gene cloning. Expression of foreign genes. Analysing genes and gene expression. Analysing whole genomes. Molecular biotechnology and applications. Immunochemical techniques. Production of antibodies. Purification and fragmentation of immunoglobulins. Immunoprecipitation. Labelling antibodies. Immunoblotting. Immunoassays. Immunohisto/ cytochemistry. Affinity and avidity. Immunochemical use of surface plasmon resonance. Centrifugation techniques. Basic principles of sedimentation. Centrifuges and their uses. Design and care of preparative rotors. Sample containers. Separation methods in Preparative ultracentrifuges. Performing density gradient separations. Selection, efficiency and applications of preparative rotors. Analysis of subcellular fractions. Some applications of the analytical ultracentrifuge. Safety aspects in the use of centrifuges. Protein structure, purification and characterisation. Ionic properties of amino acids and proteins. Protein structure. Protein purification. Protein structure determination. Biomolecular interactions: Enzymes. Receptor-ligand binding. Enzymes: characteristics and nomenclature. Enzyme steady-state kinetics. Enzyme assays. Substrate assays. Enzyme pre-steady-state kinetics. Enzyme active sites and catalytic mechanisms. Immobilised enzymes. Cellular control of metabolic activity. Biomolecular interactions: Cell surface receptors and transporters. Cell surface receptor classification. Quantitative aspects pf receptor-ligand binding. Receptor structures. Mechanisms of signal transduction. Signal amplification. Membrane transport process. Physical diffusion. Facilitated transport. Active transport and ion channels. Receptor-mediated endocytosis. Spectroscopic techniques: Atomic and molecular electronic spectroscopy. Y-ray spectroscopy and Y-ray resonance spectroscopy. X-ray spectroscopy. Ultraviolet and visible light spectroscopy. Spectrofluorimetry. Circular dichroism spectroscopy. Turbidimetry and nephelometry. Luminometry. Atomic spectroscopy. Lasers. Spectroscopic techniques: Vibrational spectroscopy and electron and nuclear spin orientation in magnetic fields. Infrared and Raman spectroscopy. Electron spin resonance spectroscopy. Nuclear magnetic resonance spectroscopy. Mass spectrometric techniques. Mass spectrometer. Electron impact ionisation. Chemical ionisation. Field ionisation. Ion desorption methods. Ion evaporation methods. Analysers. Detectors. Tandem mass spectrometry. Electrophoretic techniques. Support media. Electrophoresis of proteins. Electrophoresis of nucleic acids. Capillary electrophoresis. Chromatographic techniques. Chromatography theory and practice. Low pressure column chromatography. Adsorption chromatography. Partition chromatography. Ion-exchange chromatography. Molecular exclusion (permeation) chromatography. Affinity chromatography. Gas-liquid chromatography. Thin-layer (planar) chromatography. Selection of a chromatographic system. Radioisotope techniques. Nature of radioactivity. Detection and measurement of radioactivity. Other practical aspects of counting radioactivity and analysis of data. Inherent advantages and restrictions of radiotracer experiments. Safety aspects. Applications of radioisotopes in the biological sciences. Electrochemical techniques. Principles of electrochemical techniques. Redox reactions. The pH electrode. Ion-selective and gas-sensing electrodes. The Clark oxygen electrode. Electrochemical detectors for HPLC. Biosensors