Introduction to Experimental Biophysics

Biological Methods for Physical Scientists

Jay L. Nadeau

• Publisher: CRC Press
• Year: 2017
• Print ISBN: 9781138088153
• eBook ISBN: 9781351000925
• Topic: Medicine
• Subtopic: Biotechnology in Medicine

Praise for the First Edition

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

"an ambitious text…. Each chapter contains protocols and the conceptual reasoning behind them, which is often useful to physicists performing biological experiments for the first time." –Physics Today

This fully updated and expanded text is the best starting point for any student or researcher in the physical sciences to gain firm grounding in the techniques employed in molecular biophysics and quantitative biology. It includes brand new chapters on gene expression techniques, advanced techniques in biological light microscopy (super-resolution, two-photon, and fluorescence lifetime imaging), holography, and gold nanoparticles used in medicine. The author shares invaluable practical tips and insider's knowledge to simplify potentially confusing techniques. The reader is guided through easy-to-follow examples carried out from start to finish with practical tips and insider's knowledge. The emphasis is on building comfort with getting hands "wet" with basic methods and finally understanding when and how to apply or adapt them to address different questions.

Jay L. Nadeau is a scientific researcher and head of the Biomedical Engineering in Advanced Applications of Quantum, Oscillatory, and Nanotechnological Systems (BEAAQONS) lab at Caltech and was previously associate professor of biomedical engineering and physics at McGill University.

1Introduction and Background

1.1Basic Biochemistry

Molecules important to molecular biophysics

The chemicals of life are organic compounds, or compounds that contain carbon. Carbon (C, atomic number 6) is one of the few tetravalent atoms, meaning that it has four valence electrons available to form bonds with other atoms. Each of the four atoms to which it bonds can be different and can include other carbons. Carbon is thus central to the formation of complex, three-dimensional molecules, and it makes up about 10.7% of the atomic ratio of living matter. Other molecules necessary for the building blocks of life are hydrogen (H, atomic number 1, monovalent, 60.5%); oxygen (O, atomic number 8, divalent, 25.7%); nitrogen (atomic number 7, trivalent, 2.4%); phosphorus (P, atomic number 15, trivalent up to hexavalent, 0.17%); and sulfur (S, atomic number 16, divalent, tetravalent, or hexavalent, 0.13%).

The valence of the key elements forms the basis of the structural model of organic chemistry that permits us to predict which combinations of atoms will combine to form stable molecules. Figure 1.1 shows the classes of organic molecules that are most important in biochemistry and their functional groups. If the letter R is used to designate any chemical moiety besides hydrogen, then an amine has the general formula RNH2 (for a primary amine), R2NH (for a secondary amine), or R3N (for a tertiary amine). A carboxylic acid is RCOOH; at physiological pH, it will usually dissociate into a free proton (H+) and a negatively charged ion RCOO− (called a carboxylate). A ketone is RCOR where the second R is not an OH group. Phosphates in biology have the form ${\text{RPO}}_3^{2-}$

; when R is OH, this is referred to as inorganic phosphate or Pi. Alcohols are ROH where R can be nearly anything; any biomolecule with a name ending in -ol terminates in an OH group. A sulfhydryl, also known as a thiol group, is RSH. Thiols are also known as mercaptans. Finally, an aromatic group is a planar ring that may be made of carbon only or of carbon plus oxygen, nitrogen, or sulfur (called heterocyclic compounds). The simplest example is the six-carbon benzene ring.

Figure 1.1Functional groups seen in biochemistry.

The structural and functional makeup of a cell results from combinations of four basic molecular types, each of which falls into one or more of the categories in Figure 1.1; these molecules join end to end (polymerize) to achieve their final active form:

• Amino acids (polymerize to form peptides and proteins). There are twenty naturally occurring amino acids, whose structure consists of a central carbon atom with a carboxylic acid on one end and a primary amine on the other, and a side chain that branches off the first carbon after the amine. The side chain determines the amino acid’s identity and ranges from a hydrogen (glycine) to complex charged or aromatic groups (Figure 1.2). Short chains of amino acids are called peptides and may be synthesized by organisms like fungi in order to kill bacteria. The example shown is bacitracin, which is a cyclic peptide active against many bacteria; it is often found in first-aid creams. Some peptides are available from biological suppliers, and custom peptides are also available, though costly. Full-length proteins are encoded genetically and synthesized as a long polypeptide chain. They then fold to form their final tertiary structure; the example shown is green fluorescent protein, or GFP, which has 238 amino acids. The physics of protein folding still remains largely a mystery. Proteins usually cannot be purchased but must be expressed and...

Table of contents

• Cover
• Half Title Page
• Series Page
• Title Page
• Copyright Page
• Contents
• Series Preface
• Preface
• Acknowledgments
• Author
• Contributors
• Chapter 1 Introduction and Background
• Chapter 2 Basic Molecular Cloning of DNA and RNA
• Chapter 3 Expression of Genes in Bacteria, Yeast, and Cultured Mammalian Cells
• Chapter 4 Advanced Topics in Molecular Biology
• Chapter 5 Protein Expression Methods
• Chapter 6 Protein Crystallization
• Chapter 7 Introduction to Biological Light Microscopy
• Chapter 8 Advanced Light Microscopy Techniques
• Chapter 9 Advanced Topics in Microscopy II: Holographic Microscopy
• Chapter 10 Quantitative Cell Culture Techniques
• Chapter 11 Semiconductor Nanoparticles (Quantum Dots)
• Chapter 12 Gold Nanoparticles
• Chapter 13 Advanced Topics in Gold Nanoparticles: Biomedical Applications
• Chapter 14 Surface Functionalization Techniques
• Chapter 15 Electrophysiology
• Chapter 16 Spectroscopy Tools and Techniques
• Chapter 17 Introduction to Nanofabrication
• Glossary
• Appendix A Common Solutions
• Appendix B Common Media
• Appendix C Restriction Endonucleases
• Appendix D Common Enzymes
• Appendix E Fluorescent Dyes and Quenchers
• Appendix F Fluorescent Proteins
• Index