Introduction to Experimental Biophysics

Introduction to Experimental Biophysics: Biological Methods for Physical Scientists

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Features

    • Gives all the necessary practical tools and conceptual background needed for those with no experience in experimental biology
    • Firm footing in basic biology terminology and concepts, giving a clear understanding of what is experimentally feasible, then building gradually to more advanced techniques
    • Begins with an introduction to the building blocks of life-genes and proteins-and then provides practical guidelines on handling these molecules, from purification to amplification to analysis, then builds gradually from these foundations to more advanced topics

    Summary

    Increasing numbers of physicists, chemists, and mathematicians are moving into biology, reading literature across disciplines, and mastering novel biochemical concepts. To succeed in this transition, researchers must understand on a practical level what is experimentally feasible. The number of experimental techniques in biology is vast and often specific to particular subject areas; nonetheless, there are a few basic methods that provide a conceptual underpinning for broad application. Introduction to Experimental Biophysics is the ideal benchtop companion for physical scientists interested in getting their hands wet.

    Assuming familiarity with basic physics and the scientific method but no previous background in biology or chemistry, this book provides:

      • A thorough description of modern experimental and analytical techniques used in biological and biophysical research
      • Practical information and step-by-step guidance on instrumentation and experimental design
      • Recipes for common solutions and media, lists of important reagents, and a glossary of biological terms used

      Developed for graduate students in biomedical engineering, physics, chemical engineering, chemistry, mathematics, and computer science, Introduction to Experimental Biophysics is an essential resource for scientists to overcoming conceptual and technical barriers to working in a biology wet lab.

      Table of Contents

      Introduction and Background
      Basic Biochemistry
      Energies and Potentials
      Principles of Spectroscopy
      Cells
      DNA, RNA, Replication, and Transcription
      Translation and the Genetic Code
      Protein Folding and Trafficking
      Alternative Genetics
      What Is Cloning?
      Design of a Molecular Biology Experiment and How to Use This Book
      Questions and Problems
      Background Reading

      Molecular Cloning of DNA and RNA
      Introduction
      Obtaining and Storing Plasmids
      Selection of an Appropriate E. coli Amplification Strain: Transformation of E. coli with Plasmid
      Plasmid Amplification and Purification
      Plasmid Restriction Mapping and Agarose Gel Electrophoresis
      An Example of Cloning Experiment
      Cloning by the Polymerase Chain Reaction
      Sequencing
      RNA Methods
      Southern and Northern Blots
      Solutions for Large Cloning Problems and Multiple Inserts
      Mutagenesis and Directed Evolution
      Microarrays
      Summary
      Questions and Problems
      Background Reading

      Expression of Genes in Bacteria, Yeast, and Cultured Mammalian Cells
      Introduction
      Expressing Genes in Microorganisms
      Mammalian Cell Culture
      Transfection of Mammalian Cells I: Standard Techniques
      Transfection of Mammalian Cells II: Specialized Physical Methods for Special Occasions
      Transfection of Mammalian Cells III: Viruses
      Summary
      Questions and Problems
      Background Reading

      Protein Expression Methods
      Introduction
      Expression Systems
      Identification of a DNA Source
      Selecting an Expression Vector
      Subcloning into an Expression Vector
      Selection of an Expression Strain or Cell Line
      Protein Expression
      Checking Protein Expression (and Purity) Using SDS-PAGE
      Protein Isolation and Purification
      Chromatography
      Buffer Exchange and Concentration
      Example Experiment: Expression and Purification of Fluorescent Protein Dronpa
      Conclusions and Final Remarks
      Background Reading

      Protein Crystallization
      Introduction
      Crystallization of Macromolecules
      Preparation of Proteins for Crystallization
      Components of Crystallization Solutions
      Other Factors Affecting Crystallization
      Crystallization Strategies
      Example Experiment: Lysozyme
      Data Collection and Structure Determination Using X-Ray Crystallography
      A Special Case: Membrane Proteins
      Troubleshooting Q&A
      Conclusions and Final Remarks
      Questions and Problems
      Background Reading

      Introduction to Biological Light Microscopy
      Introduction
      The Physics of Microscopy: Magnification and Resolution
      Anatomy of a Biological Microscope
      Brightfield Imaging Techniques
      Basic Fluorescence Microscopy
      Fluorophores for Cell Labeling
      Fluorescent Proteins
      Multispectral Imaging Using Acousto-Optical Tunable Filters
      Advanced Techniques
      Summary and Remarks
      Questions and Problems
      Background Reading

      Quantitative Cell Culture Techniques
      Introduction
      Quantifying Bacterial Growth and Death
      Quantifying Mammalian Cells
      Flow Cytometry
      Example Experiment: Determining Leukemic B-Cells and T-Cells by Flow Cytometry
      Quantifying Viruses
      Measuring Cell Populations Using Quantitative PCR
      Summary and Final Remarks
      Questions and Problems
      Background Reading

      Semiconductor Nanoparticles (Quantum Dots)
      Introduction
      Quantum Dot Properties and Synthesis
      QD Applications
      Example Experiment: Conjugation of Quantum Dots to Dopamine and Quantifying the Effects on Fluorescence per Molecule Bound
      Summary and Remarks
      Questions and Problems
      Background Reading

      Gold Nanoparticles
      Introduction
      The Physics of Scattering and Spherical Metal Nanoparticles
      Synthesis of Gold Nanoparticles
      Characterization and Surface Modification of Gold Nanoparticles
      Applications for Colorimetric Detection and Microscopy
      Sample Experiment: Labeling Cells with Lectin-Tagged Au Nanoparticles
      Applications in Surface-Enhanced Raman Scattering
      Gold Nanoparticles as Photothermal Transducers
      Conclusion
      Questions and Problems
      Background Reading

      Surface Functionalization Techniques
      Introduction
      Preparing Monolayers Using Functional Silanes or Thiols
      Techniques for Characterizing Surface Monolayers
      Functionalization of Modified Surfaces Using Cross-Linkers
      Example Experiment: Preparing a Silane–Biotin–Streptavidin Sandwich on SiO2 Features on an Si Chip
      Preventing Nonspecific Binding of Biomolecules
      Assembling Membrane Proteins on Surfaces
      Testing the Function of Immobilized Proteins
      Conclusion and Final Remarks
      Questions and Problems
      Background Reading

      Electrophysiology
      Introduction
      Physical Basis and Circuit Models
      Solutions and Blockers
      Instrumentation
      Lipid Bilayer Setup
      Cell Patch-Clamp Setup: What Is Needed?
      The Art and Magic of Pipette Pulling
      Step-by-Step Guide to Perform a Whole-Cell Recording
      Example Experiment: Whole-Cell Recording on Cells
      A Brief Introduction to Single-Channel Modeling and Data Analysis
      Network Recording
      Conclusions and Final Remarks
      Questions and Problems
      Background Reading

      Spectroscopy Tools and Techniques
      Introduction
      Guiding Principles
      UV–Vis Absorbance Spectroscopy
      Fluorescence Spectroscopy
      Time-Resolved Emission
      Time-Resolved Absorption
      Infrared Spectroscopy
      Nuclear Magnetic Resonance
      Electron Paramagnetic Resonance Spectroscopy
      X-Ray Spectroscopy
      Example Experiment: Characterization of CdSe/ZnS Nanoparticle Bioconjugate Using UV–Vis, Fluorescence Emission, Time-Resolved Emission, FTIR, and EPR Spectroscopy
      Final Comments
      Questions and Problems
      Background Reading

      Appendix
      Glossary
      Index

      Author Bio(s)

      Jay L. Nadeau is an associate professor of biomedical engineering and physics at McGill University (2004–present). Her research interests include nanoparticles, fluorescence imaging, and development of instrumentation for the detection of life elsewhere in the solar system.

      She has published over 50 papers on topics ranging from theoretical condensed matter physics to experimental neurobiology to the development of anticancer drugs and, in the process, has used almost every technique described in this book. Her work has been featured in New Scientist, Highlights in Chemical Biology, Radio Canada’s Les Années Lumière, Le Guide des Tendances, and in educational displays in schools and museums. Her research group features

      chemists, microbiologists, roboticists, physicists, and physician-scientists, all learning from each other and hoping to speak each other’s language. A believer in bringing biology to physicists as well as physics to biologists, she has created two graduate-level courses: methods in molecular biology for physical scientists and mathematical cellular physiology. She also teaches pharmacology in the medical school and was one of the pioneers in the establishment of multiple mini-interviews for medical school admission.

      She has an adjunct position with The Jackson Laboratory in Bar Harbor, Maine, and collaborators in industry and academia in the United States, Europe, Australia, and

      Japan. She has given several dozen invited talks at meetings of the American Chemical Society, American Geophysical Union, the International Society for Optics and Photonics (SPIE), the Committee on Space Research, and many others. Before McGill, she was a member of the Jet Propulsion Laboratory’s Center for Life Detection, and previous to that a Burroughs-Wellcome postdoctoral scholar in the laboratory of Henry A. Lester at Caltech. She received her PhD in physics from the University of Minnesota in 1996.

      Editorial Reviews

      This book is essential reading for any physical scientist who is interested in performing biological research.
      Contemporary Physics

      … an ambitious text aimed at educating new graduate students about the important and most common techniques used in a modern biological physics laboratory; it could also serve nicely as a reference manual for advanced graduate students of new or underused protocols. … Overall, the many outstanding qualities should make it an essential part of the biophysicist’s collection.
      —Jennifer L. Ross, Physics Today, August 2012

      Very useful as a resource to get a basic understanding of methodology outside one's realm of expertise … very readable.
      —Gary F. Polking, Ph.D., Iowa State University

      The book provides a comprehensive overview of diverse methods in biophysics. It will be a great resource for every working scientist in the physical sciences. It would also be a great supporting text to read as part of an introductory course in biophysical methods, particularly for graduate students and postdocs entering the field from other disciplines.
      —Anthony J. Koleske, Yale University

      This book provides a broad overview on the many interrelated disciplines shaping modern biophysical research. Its structure evolves from the basics of biochemistry through the principles of relevant analytical techniques to the chemistry of nanoparticles and surfaces. The many chapters appear to be rather exhaustive, clearly organized and beautifully illustrated. I believe that this book will be a useful tool to undergraduate and graduate students and a valuable reference for researchers in the field.
      —Françisco M. Raymo, University of Miami

      This book fills the need for a practical, hands-on guide for physical scientists who are moving into biological research.
      —Daniel A. Beard, Medical College of Wisconsin

      As scientists from more quantitative fields expand further into molecular and cellular biology, their labs need to acquire new biological methods for sample preparation and handling. These skills are not traditionally available to physicists and chemists. This book will be appropriate for any experimentalist in chemistry or physics who is moving into biological work. It will also be excellent reading material for undergraduate or graduate students who will be working in a biologically oriented lab, as well as for an advanced lab class in biophysics or bioengineering.
      —Mark C. Williams, Northeastern University

      This book will be very useful for training the growing number of researchers and students from physical sciences to become more familiar with techniques used in biology. The author has made a great effort to keep everything defined and simple.
      —James A. Forrest, Department of Physics and Associate Dean of Research, Faculty of Science, University of Waterloo

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