Electrophoresis in Practice is a laboratory guide for successful electrophoretic separations and is divided into two parts to provide readers with a thorough presentation of the fundamentals followed by a detailed description of the most common methods currently in use.

This New Edition Features:

• A section on difference gel electrophoresis
• The chapter on proteome analysis is practically all new and considerably extended
• New attractive layout
• Around 10 % new literature references.

Contents

1. Fundamentals
   1. Electrophoresis
      • 1.0 General
      • 1.1 Electrophoresis in non-restrictive gels
      • 1.2 Electrophoresis in restrictive gels

   2. Isotachophoresis
      • 2.1 Migration with the same speed
      • 2.2 “Ion train” separation
      • 2.3 Zone sharpening effect
      • 2.4 Concentration regulation effect

   3. Isoelectric focusing
      • 3.1 Principles
      • 3.2 Gels for IEF
      • 3.3 Temperature
      • 3.4 Controlling the pH gradient
      • 3.5 The kinds of pH gradients
      • 3.6 Protein detection in IEFgels
      • 3.7 Preparative isoelectric focusing
      • 3.8 Titration curve analysis

   4. Blotting
      • 4.1 Principle
      • 4.2 Transfer methods
      • 4.3 Blotting membranes
      • 4.4 Buffers for electrophoretic transfers
      • 4.5 General staining
      • 4.6 Blocking
      • 4.7 Specific detection
      • 4.8 Protein sequencing
      • 4.9 Transfer problems

   5. Interpretation of electropherograms
      • 5.1 Introduction
      • 5.2 Image analysis

   6. Proteome Analysis
      • 6.1 General
      • 6.2 Sample preparation
      • 6.3 Two-dimensional electrophoresis.
      • 6.4 Detection techniques
      • 6.5 Image analysis
      • 6.6 Protein spot identification
      • 6.7 Bioinformatics
      • 6.8 Functional proteomics

   7. Instrumentation
      • 7.1 Current and voltage conditions
      • 7.2 Power supply
      • 7.3 Separation chambers
      • 7.4 Staining apparatus for gels and blots
      • 7.5 Automated electrophoresis
      • 7.6 Instruments for 2-D electrophoresis
      • 7.7 Safety measures
      • 7.8 Environmental aspects

2. Equipment and Methods
   • Equipment for Part II
     • Instrumentation
     • Special laboratory equipment
     • Consumables
     • Chemicals

   • Method 1: PAGE of dyes
     • Sample preparation
     • Stock solutions
     • Preparing the casting cassette
     • Casting ultrathin-layer gels
     • Electrophoretic separation

   • Method 2: Agarose and immuno electrophoresis
     • Sample preparation
     • Stock solutions
     • Preparing the gels
     • Electrophoresis
     • Protein detection

   • Method 3: Titration curve analysis
     • Sample preparation
     • Stock solutions
     • Preparing the blank gels
     • Titration curve analysis
     • Coomassie and silver staining
     • Interpreting the curves

   • Method 4: Native PAGE in amphoteric buffers
     • Sample preparation
     • Stock solutions
     • Preparing the empty gels
     • Electrophoresis
     • Coomassie and silver staining

   • Method 5: Agarose IEF
     • Sample preparation
     • Preparing the agarose gel
     • Isoelectric focusing
     • Protein detection

   • Method 6: PAGIEF in rehydrated gels
     • Sample preparation
     • Stock solutions
     • Preparing the blank gels
     • Isoelectric focusing
     • Coomassie and silver staining
     • Perspectives

   • Method 7: Horizontal SDS-PAGE
     • Sample preparation
     • Stock solutions for gel preparation
     • Preparing the casting cassette
     • Gradient gel
     • Electrophoresis
     • Protein detection
     • Blotting
     • Perspectives

   • Method 8: Vertical PAGE
     • Sample preparation
     • Stock solutions
     • Single gel casting
     • Multiple gel casting
     • Electrophoresis
     • SDS electrophoresis of small peptides
     • Two-dimensional electrophoresis
     • DNA electrophoresis
     • Long shelflife gels
     • Detection of bands

   • Method 9: Semi-dry blotting of proteins
     • Transfer buffers
     • Technical procedure
     • Staining of blotting membranes

   • Method 10: IEF in immobilized pH gradients
     • Sample preparation
     • Stock solutions
     • Immobiline recipes
     • Preparing the casting cassette
     • Preparing the pH gradient gels
     • Isoelectric focusing
     • Staining
     • Strategies for IPG focusing

   • Method 11: High-resolution 2-D electrophoresis
     • Sample preparation
     • Stock solutions
     • Preparing the gels
     • Separation conditions
     • Staining procedures

   • Method 12: PAGE of double stranded DNA
     • Stock solutions
     • Preparing the gels
     • Sample preparation
     • Electrophoresis
     • Silver staining

   • Method 13: Native PAGE of single stranded DNA
     • Sample treatment
     • Gel properties
     • Buffers and additives
     • Conditions for electrophoresis
     • Strategy for SSCP analysis

   • Method 14: Denaturing gradient gel electrophoresis
     • Sample preparation
     • Rehydration solutions
     • Preparing the rehydration cassette
     • Rehydration
     • Electrophoresis
     • Silver staining

   • Method 15: Denaturing PAGE of DNA
     • Sample preparation
     • Solutions
     • Rehydration
     • Electrophoresis
     • Silver staining

   • Appendix Trouble-shooting
     • Isoelectric focusing
     • PAGIEFwith carrier ampholytes
     • Agarose IEFwith carrier ampholytes
     • Immobilized pH gradients
     • SDS electrophoresis
     • Vertical PAGE
     • Semidry blotting
     • 2-D electrophoresis
     • DNA electrophoresis

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