Electrophoresis in Practice

A Guide to Methods and Applications of DNA and Protein Separations

Reiner Westermeier

• Publisher: Wiley-VCH
• Year: 2016
• Print ISBN:
  9783527338801
• eBook ISBN:
  9783527695195
• Edition: 5
• Topic: Ciencias biológicas
• Subtopic: Genética y genómica

This fifth edition of the successful, long-selling classic has been completely revised and expanded, omitting some topics on obsolete DNA electrophoresis, but now with a completely new section on electrophoretic micro-methods and on-the-chip electrophoresis.
The text is geared towards advanced students and professionals and contains extended background sections, protocols and a trouble-shooting section. It is now also backed by a supplementary website providing all the figures for teaching purposes, as well as a selection of animated figures tested in many workshops to explain the underlying principles of the different electrophoretic methods.

Part I
Fundamentals

Introduction

Electrophoresis is besides chromatography the most frequently applied separation technique for the analysis of protein, glycan, and nucleic acid mixtures. With electrophoresis high separation efficiency can be achieved using a relatively simple setup of equipment. It is mainly applied for analytical rather than for preparative purposes. However, with the advent of amplification of DNA fragments with polymerase chain reaction (PCR®), and highly sensitive and powerful mass spectrometry (MS) analysis of proteins and peptides, so called “analytical amounts” of electrophoretically separated fractions can be further analyzed.

The main fields of application are biological and biochemical research, protein chemistry, pharmacology, forensic medicine, clinical investigations, veterinary science, food control as well as molecular biology. The monograph by Andrews (1986) is one of the most complete and practice-oriented books about electrophoretic methods. In the present book, electrophoretic methods and their applications will be presented in a much more condensed form.

Principle

Under the influence of an electrical field charged molecules or particles migrate into the direction of the electrode bearing the opposite charge. During this process, the substances are in aqueous solution. Because of their varying charges and masses, different molecules and particles of a mixture will migrate at different velocities and will thus be separated into single fractions.

The electrophoretic mobility, which is a measure of the migration velocity, is a significant and characteristic parameter of a charged molecule or particle. It is dependent on the pK values of the charged groups and the size of the molecule or particle. It is influenced by the type, concentration and pH of the buffer, by the temperature as well as by the nature of the supporting material. Electrophoretic separations are carried out in free solutions – like in capillary, microchip, and free flow systems – or in stabilizing media such as thin-layer plates, films, or gels. Detailed theoretical explanations can be found in the textbook edited by Lottspeich and Engels (2016).

Sometimes the relative electrophoretic mobility of substances is specified. It is calculated relative to the migration distance of a standard substance, mostly a dye like bromophenol blue, which has been applied as an internal standard. The relative mobility is abbreviated as m_r or R_m.

Three basically different electrophoretic separation methods are performed in practice nowadays:

1. Electrophoresis, sometimes called Zone Electrophoresis (ZE)
2. Isotachophoresis (ITP)
3. Isoelectric focusing (IEF).

The three separation principles are illustrated in Figure p1.1. There is a fourth method: “moving boundary electrophoresis,” which is described below. However this technique has no practical importance anymore.

1. In ZE a homogeneous buffer system is used over the whole separation time and range so as to ensure a constant pH value. The migration distances during a defined time limit are a measure of the electrophoretic mobilities of the various substances. It can be applied to nonamphoteric as well as amphoteric molecules. During the separation diffusion can lead to blurred zones, which reduces the sensitivity of detection and the resolution. Buffer reservoirs at the anodal and the cathodal side are needed to maintain the buffer conditions during the separation.
2. In ITP, the separation is carried out in a discontinuous buffer system. The ionized sample migrates between a leading electrolyte with a high mobility and a terminating – sometimes called trailing – ion with a low mobility, all of them migrating with the same speed. The different components are separated according to their electrophoretic mobilities and form stacks: the substance with the highest mobility follows directly the leading ion, the one with the lowest mobility migrates directly in front of the terminating electrolyte. In ITP there is a concentration effect which works against diffusion. In comparison to other electrophoretic and chromatographic separation methods, ITP is considered exotic, because there are no spaces between the zones, and the bands are not “peaks” (Gaussian curves) but “spikes” (concentration dependent bands). ITP is mostly applied for stacking of the samples during the first phase of disc electrophoresis.
3. IEF takes place in a pH gradient and can only be used for amphoteric substances such as peptides and proteins. The charged molecules move toward the anode or the cathode until they reach a position in the pH gradient where their net charges are zero. This pH value is the “isoelectric point” (pI) of the substance. Since the molecule is no longer charged, the electric field does not have any influence on it. Should the substance diffuse away, it will gain a net charge again, and the applied electric field will cause it to migrate back to its pI. This concentrating effect leads to the name focusing. Thus also with IEF there is no problem with diffusion.

Figure p1.1 The three electrophoretic separation principles. Explanations in the text. A and B are the components of the sample.

Areas of Applications

Mainly proteins, glycans, peptides, and nucleic acids are separated. Electrophoretic methods are used for the qualitative characterization of a substance or mixture of substances, for control of purity, quantitative determinations, and preparative purposes. The most prominent fields are the Genome and the Proteome analysis. The word “Proteome” was introduced by Mark Wilkins during a congress in Sienna 1994, in writing in the publication by Wasinger et al. (1995) 1 year later.

The scope of the applications ranges from whole cells and particles to nucleic acids, proteins, peptides, amino acids, glycans, organic acids and bases, drugs, pesticides, and inorganic anions and cations – in short – every particle or molecule that can carry a charge.

The Sample

An important criterion for the choice of the appropriate electrophoretic method is the nature of the sample to be analyzed. There must be no solid particles or fatty components suspended in the solution. Those interfere with the separation by blocking the pores of the matrix. Sample solutions are mostly centrifuged – sometimes also desalted – before electrophoresis.

Substances which ...

Table of contents

• Cover
• Title Page
• Copyright
• Table of Contents
• Foreword
• Abbreviations, Symbols, Units
• Preface
• Part I: Fundamentals
• Part II: Equipment and Methods
• Method 1: PAGE of Dyes
• Method 2: Agarose and Immunoelectrophoresis
• Method 3: Titration Curve Analysis
• Method 4: Native PAGE in Amphoteric-Buffers
• Method 5: Agarose IEF
• Method 6: PAGIEF in Rehydrated Gels
• Method 7: Horizontal SDS-PAGE
• Method 8: Vertical PAGE
• Method 9: Semidry Blotting of Proteins
• Method 10: IEF in Immobilized pH Gradients
• Method 11: High-Resolution 2D Electrophoresis
• Method 12: PAGE of DNA Fragments
• Appendix: Troubleshooting
• Index
• End User License Agreement