Introduction to Experimental Biophysics: Biological Methods for Physical Scientists
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
