Introduction to Experimental Biophysics Biological Methods for Physical Scientists

About this book

A practical companion for physical scientists investigating wet laboratory experiments and biological research areas, this book gives a clear understanding of what is experimentally feasible. Introducing basic terminology and concepts, the text begins with simple, fundamental methods and builds gradually to more advanced techniques.

Organized into three main sections addressing genes, proteins, and expression systems, the book employs a straight-forward structure allowing readers to follow the process involved in the production of nucleic acids and proteins and the different levels of purification required for each type of analysis.

Contents

Introduction and Background Basic biochemistry Energies and potentials Spectroscopy methods Cells DNA, RNA, transcription, and reverse transcription Translation, protein folding and trafficking What is cloning? Design of a molecular biology experiment DNA and RNA Methods Obtaining and storing DNA and RNA vectors Expression, amplification, and purification of DNA and RNA Cloning by restriction enzyme digestion and PCR; software tools Sequencing Cosmids, A vectors, bacterial artificial chromosomes Mutagenesis Directed evolution Protein Expression Methods Protein expression in bacteria Protein expression in yeast and insect cells Purification of soluble proteins Purification of membrane-bound proteins Protein Characterization Methods Crystallization in 2-D and 3-D X-ray diffraction Electron diffraction Nuclear magnetic resonance Other methods Introduction to Light Microscopy Basic concepts Wavelengths and filters Laser excitation Brightfield, phase contrast, and stains Advanced Microscopy Techniques Fluorescence microscopy Confocal and two-photon microscopy Electron microscopy Atomic force microscopy Expression Systems Basics of tissue culture Primary cells Lipid bilayer techniques Microbial expression systems Quantitative Tissue Culture Techniques Growth curves and live/dead stains Assays for cell growth and metabolism (MTT, XRB) Reactive oxygen species assays Fluorescence-based assays Surface Functionalization Protein immobilization Tests for function of immobilized proteins Tests for nonspecific binding Electrophysiology Single-cell methods Two- and three-cell methods Multielectrode arrays Optical recording from networks Advanced Cell-Labeling Techniques Membrane dyes Organelle-specific dyes Ion-specific dyes Ratiometric imaging Labels for electron microscopy Nanoparticle Methods Fluorescent nanoparticles (quantum dots): principles and synthesis Synthesis of gold nanoparticles Cell labeling with quantum dots: surface labeling Uptake of quantum dots Functionalization of nanogold and cellular labeling Principles of light microscopy using quantum dots and nanogold Principles of electron microscopy using quantum dots and nanogold Advanced Fluorescence Methods Fluorescence recovery after photobleaching (FRAP) Total internal reflection (TIRF) Fluorescence lifetime imaging (FLIM) Fluorescence photo-activation localization microscopy (FPALM) Spectroscopy Tools and Techniques Fluorescence spectroscopy UV-Vis and IR spectroscopy Time-resolved absorbance techniques Time-resolved emission techniques Electron paramagnetic resonance (EPR) spectroscopy Appendix 1: Quantitative Data Analysis Experimental design Statistics and significance tests Error propagation Single-particle and single-channel mathematics Appendix 2: Useful Lab Data Codon tables Recipes for common solutions (phosphate buffered saline, Tris, etc.) Common media for bacteria and cells Restriction enzymes Commonly used enzymes for DNA and RNA manipulations Dye and fluorescent protein excitation/emission

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Biography

Jay Nadeau, Department of Biomedical Engineering, McGill University, Montreal, Quebec, Canada