Introduction to Experimental Biophysics

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.

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