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Electrophoresis in Practice, 4th, Revised and Updated Edition

ISBN: 978-3-527-31181-1
426 pages
March 2005, Wiley-Blackwell
Electrophoresis in Practice, 4th, Revised and Updated Edition (3527311815) cover image
This laboratory guide for successful electrophoretic separations 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 fourth edition retains the successful concept of its predecessors, yet features a brand-new layout, and is further enhanced by a section on difference gel electrophoresis, while the chapter on proteome analysis is practically all new and considerably extended, plus there are now around 10 % new literature references.
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Foreword.

Preface.

Abbreviations.

Part I Fundamentals.

Introduction.

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.

Part II Equipment and Methods.

Equipment for Part II.

Instrumentation.

Special laboratory equipment.

Consumables.

Chemicals.

Method 1: PAGE of dyes.

1 Sample preparation.

2 Stock solutions.

3 Preparing the casting cassette.

4 Casting ultrathin-layer gels.

5 Electrophoretic separation.

Method 2: Agarose and immuno electrophoresis.

1 Sample preparation.

2 Stock solutions.

3 Preparing the gels.

4 Electrophoresis.

5 Protein detection.

Method 3: Titration curve analysis.

1 Sample preparation.

2 Stock solutions.

3 Preparing the blank gels.

4 Titration curve analysis.

5 Coomassie and silver staining.

6 Interpreting the curves.

Method 4: Native PAGE in amphoteric buffers.

1 Sample preparation.

2 Stock solutions.

3 Preparing the empty gels.

4 Electrophoresis.

5 Coomassie and silver staining.

Method 5: Agarose IEF.

1 Sample preparation.

2 Preparing the agarose gel.

3 Isoelectric focusing.

4 Protein detection.

Method 6: PAGIEF in rehydrated gels.

1 Sample preparation.

2 Stock solutions.

3 Preparing the blank gels.

4 Isoelectric focusing.

5 Coomassie and silver staining.

6 Perspectives.

Method 7: Horizontal SDS-PAGE.

1 Sample preparation.

2 Stock solutions for gel preparation.

3 Preparing the casting cassette.

4 Gradient gel.

5 Electrophoresis.

6 Protein detection.

7 Blotting.

8 Perspectives.

Method 8: Vertical PAGE.

1 Sample preparation.

2 Stock solutions.

3 Single gel casting.

4 Multiple gel casting.

5 Electrophoresis.

6 SDS electrophoresis of small peptides.

7 Two-dimensional electrophoresis.

8 DNA electrophoresis.

9 Long shelflife gels.

10 Detection of bands.

Method 9: Semi-dry blotting of proteins.

1 Transfer buffers.

2 Technical procedure.

3 Staining of blotting membranes.

Method 10: IEF in immobilized pH gradients.

1 Sample preparation.

2 Stock solutions.

3 Immobiline recipes.

4 Preparing the casting cassette.

5 Preparing the pH gradient gels.

6 Isoelectric focusing.

7 Staining.

8 Strategies for IPG focusing.

Method 11: High-resolution 2-D electrophoresis.

1 Sample preparation.

2 Stock solutions.

3 Preparing the gels.

4 Separation conditions.

5 Staining procedures.

Method 12: PAGE of double stranded DNA.

1 Stock solutions.

2 Preparing the gels.

3 Sample preparation.

4 Electrophoresis.

5 Silver staining.

Method 13: Native PAGE of single stranded DNA.

1 Sample treatment.

2 Gel properties.

3 Buffers and additives.

4 Conditions for electrophoresis.

5 Strategy for SSCP analysis.

Method 14: Denaturing gradient gel electrophoresis.

1 Sample preparation.

2 Rehydration solutions.

3 Preparing the rehydration cassette.

4 Rehydration.

5 Electrophoresis.

6 Silver staining.

Method 15: Denaturing PAGE of DNA.

1 Sample preparation.

2 Solutions.

3 Rehydration.

4 Electrophoresis.

5 Silver staining.

Appendix Trouble-shooting.

A1 Isoelectric focusing.

A1.1 PAGIEFwith carrier ampholytes.

A1.2 Agarose IEFwith carrier ampholytes.

A1.3 Immobilized pH gradients.

A2 SDS electrophoresis.

A3 Vertical PAGE.

A4 Semidry blotting.

A5 2-D electrophoresis.

A6 DNA electrophoresis.

References.

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Born in 1951, Reiner Westermeier gained his diploma in engineering in 1976, and his doctorate in 1981 at the Technical University of Munich, Germany.
He remained at the TU engaged in the development of new electrophoresis systems and applications until 1983, a period that also involved several international collaborations and lecturing tours.
From 1984 to 1987 Dr. Westermeier was employed as an electrophoresis products and applications specialist at LKB Instrument GmbH thereafter until 1990 at Pharmacia Biotech. He founded ETC Elektrophorese-Technik in 1991 for the development of new electrophoresis methods, media, and equipment. From January 1996 to July 1997 he was manager of scientific support Europe at Pharmacia Biotech in Freiburg, Germany. He has been at Amersham Pharmacia Biotech since July 1997, and its Senior Scientist for Proteomics since January 2002.
Reiner Westermeier is the author of several publications and two books, both published by WILEY-VCH.
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From the reviews of previous editions:

"The illustrations are informative and to the point...The book clearly illustrates the competence of the author. He generously shares his experience and knowledge with the reader, especially in the field of gel-based electrophoresis...an excellent reference to help the reader to become acquainted with the field of electrophoresis. The book contains theoretical and ample practical information that serves not only the practician in the electrophoresis laboratory, but also students and lecturers in electrophoretic methods." (Clinical Chemistry)

"The rigorous description of each method along with the extensive figures will easily allow the novice to reproduce these methods in the laboratory" (The Analyst)

"An excellent book which we recommend greatly and which has not to be missed in any laboratory of cellular and molecular biology which respects itself." (Cellular and Molecular Biology)

"As a comprehensive guide to the huge variety of electrophoretic methods now available, this is very good value. The superb troubleshooting appendix almost justifies the price on its own." (Laboratory Equipment Digest)
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