Protein Purification

Philip Bonner

• Publisher: Taylor & Francis
• Year: 2018
• Print ISBN: 9781138312470
• eBook ISBN: 9780429856969
• Topic: Medicine
• Subtopic: Biochemistry in Medicine

This second edition of Protein Purification provides a guide to the major chromatographic techniques, including non-affinity absorption techniques, affinity procedures, non-absorption techniques and methods for monitoring protein purity. The new edition of the book has been organized to encourage incremental learning about the topic, starting with the properties of water, progressing through the characteristics of amino acids and proteins which relate to the purification process. There is an overview of protein strategy and equipment, followed by discussions and examples of each technique and their applications.

The basic theory and simple explanations given in Protein Purification make it an ideal handbook for final year undergraduates, and postgraduates, who are conducting research projects. It will also be a useful guide to more experienced researchers who need a good overview of the techniques and products used in protein purification.

Key Features

* Guide to the major techniques used in protein purification

* Includes flowcharts to help the reader slect the best purification strategy

* Contains step-by-step protocols that guide the reader through each technique and its use

* Includes exercises and solutions

1Water, pH and Buffers

1.1Introduction

The surface of the Earth is covered in 70% water and approximately 98% of the total water is contained within the oceans, inaccessible as potable water. Fresh water comprises the remaining 2% of global water, which is predominantly locked up in the polar ice caps (1.6%), aquifers (0.36%) and lakes/rivers (0.036%). The small percentage of the total water that remains is contained within the atmosphere as clouds and living matter.

The composition of cells that make up living matter is dominated by water, which accounts for between 60–70% of the cell’s weight. The solubilising properties of water help to keep polar molecules in solution and the non-polar molecules (e.g., lipids) are driven to adopt structures that avoid contact with water. The contents of every cell, from small metabolites to large macromolecular structures like proteins, have an intimate association with water, such that proteins lack biological activity when hydrating water is not present. The movements (conformational changes) that proteins undergo during biological activity are also linked to the protein’s hydration level, with the water molecules acting as a lubricant to facilitate conformational changes. Water is also involved in an enzyme’s substrate specificity and catalytic mechanism.

Within the cell, proteins are required for a variety of tasks, including structural support, catabolic and anabolic processes, replication and cell division. To achieve these tasks proteins must fold into their structurally active conformations, with approximately 80% of the protein’s peptide groups and non-polar side chains of amino acids tightly embedded in the centre of the protein’s structure. The amino acids with polar side chains are presented on the surface of the protein, interacting with water molecules (see Chapter 2) helping to maintain the protein in solution. The water molecules associated with the surface of a protein make up the protein’s solvation (hydration) shell. This dynamic coating of the protein’s surface is approximately two layers deep, and the water molecules are constantly exchanging places with water molecules in the surrounding cell medium (bulk phase). The depth of the solvation s...

Table of contents

• Cover
• Half Title Page
• Series Page
• Title Page
• Copyright Page
• Contents
• Detailed Contents
• Preface
• Abbreviations
• Chapter 1 Water, pH and Buffers
• Chapter 2 Protein Structure and Properties
• Chapter 3 Chromatography and the Strategy of Protein Purification
• Chapter 4 The Groundwork
• Chapter 5 Non-Affinity Absorption Techniques Used to Purify Proteins
• Chapter 6 Affinity-Based Procedures Used to Purify Proteins
• Chapter 7 Non-Absorption Techniques for Purifying Proteins
• Chapter 8 Methods for Monitoring the Purity of Protein Solutions
• Appendix 1 The Laws of Thermodynamics and the Gibbs Free Energy Equation
• Appendix 2 Properties of Amino Acids
• Appendix 3 The Common Units Used in Biology
• Appendix 4 Chromatographic Runs
• Appendix 5 Determining the Concentration of a Compound in a Solution in ppm
• Appendix 6 The Answers to the Exercises in the Various Chapters
• Appendix 7 An Alphabetical List of Chromatography (C), Electrophoresis (E), Filtration Equipment (F) and Laboratory Suppliers (LS)
• Appendix 8 Buffer Tables to Prepare Buffers at a Required pH Value
• Glossary of Terms
• Index