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Introduction to Experimental Biophysics
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 expe...
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Boca Raton
CRC Press
2012
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Table of Contents:
- Introduction to the 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
- Molecular cloning of DNA and RNA. 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 and large cloning problems and multiple inserts ; Mutagenesis and directed evolution ; Microarrays ; Summary and hints
- Expression of genes in bacteria, yeast, and cultured mammalian cells. 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. Protein expression methods. Expression systems ; Identification of a DNA source ; Selecting an expression vector ; Sub-cloning into an expression vector ; Selection of an expression strain or cell line ; Protein expression ; SDS-PAGE ; Protein isolation and purification ; Chromatography ; Buffer exchange and concentration ; Example experiment : expression and purification of fluorescent protein dronpa ; Conclusions and final remarks
- Protein crystallization. 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
- Introduction to biological light microscopy. 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. Quantitative cell culture technique. 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
- Semiconductor nanoparticles (quantum dots). 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
- Gold nanoparticles. 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. Surface functionalization techniques. 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 a Si Chip ; Preventing nonspecific binding of biomolecules ; Assembling membrane proteins on surfaces ; Testing the function of immobilized proteins ; Conclusion and final remarks
- Electrophysiology. 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 performing a whole-cell recording ; Example experiment : whole-cell recording on cells transfected with K+ channels and GFP ; A brief introduction to single-channel modeling and data analysis ; Network recording ; Conclusions and final remarks
- Spectroscopy tools and techniques. 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
- Appendix. Common solutions ; Microbial growth media ; Agarose gel recipes ; Protein gel recipes ; Restriction endonucleases ; Common nucleic acide modifying enzymes ; Fluorescent dyes and quenchers ; Fluorescent proteins.