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Isoelectric Focusing

1st Edition - January 1, 1976

Editor: Nicholas Catsimpoolas

Hardback ISBN:

9 7 8 - 0 - 1 2 - 1 6 3 9 5 0 - 1

eBook ISBN:

9 7 8 - 0 - 3 2 3 - 1 5 8 0 5 - 3

Isoelectric Focusing deals with the theory and applications of isoelectric focusing. The book reviews some alternative approaches to generation of pH gradients for isoelectric… Read more

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Isoelectric Focusing deals with the theory and applications of isoelectric focusing. The book reviews some alternative approaches to generation of pH gradients for isoelectric focusing analysis, along with other relevant topics such as the theoretical aspects of steady-state isoelectric focusing, gel electrofocusing/gel electrophoresis, and transient state isoelectric focusing. This volume is organized into nine chapters and begins by looking at the history of the discovery of isoelectric focusing and correcting some blurred impressions conveyed in the literature. It then turns to a discussion of the differential equation of isoelectric focusing, the carrier ampholytes used in carrying out the technique, the use of isoelectric focusing in polyacrylamide and granulated gels as well as in free solution, and continuous-flow electrophoresis and isoelectric focusing. An attempt is made to illuminate problems in isoelectric focusing and to point out possible solutions in the areas of theory, mechanisms, ampholyte chemistry, and apparatus. Some of the processes that occur during an isoelectric focusing experiment are also considered. This book is intended for anyone who needs to know what isoelectric focusing is all about.

List of Contributors

Preface

1 Isoelectric Focusing

I. An Essential Condition for Isoelectric Focusing

II. Isoelectric Focusing—A Family of Three Effects

III. Demonstration of the Focusing and Evacuation Effects

IV. Limitations of the Prepared pH Gradient Method and the Success of the Natural pH Gradient Method

V. Some Recent Alternative Approaches to Generation of pH Gradients for Isoelectric Focusing Analysis

References

2 Theoretical Aspects of Steady-State Isoelectric Focusing

I. Introduction

II. The Differential Equation of Isoelectric Focusing

III. Convection-Free Electrolysis of a Single Strong Acid or Strong Base

IV. Convection-Free Electrolysis of a Single Weak Acid or Weak Base

V. Convection-Free Electrolysis of an Acidic or Basic Ampholyte with Only Two Protolytic Groups in the Isoelectric Region

VI. Convection-Free Electrolysis of a Mixture of Two Ampholytes

VII. Convection-Free Electrolysis of a Mixture of Many Ampholytes

References

3 The Carrier Ampholytes

I. Desirable Properties

II. Synthesis of a System of Carrier Ampholytes

III. Physicochemical Properties: Buffer Capacity and Conductivity

IV. Number of Carrier Ampholyte Species with Different pI

V. Light Absorption

VI. Determination of pi of Proteins in the Presence of Carrier Ampholytes and Other Additives

VII. Stability of Proteins and Use of Additives

VIII. Separation of Carrier Ampholytes from Proteins

IX. Possible Interaction with Proteins

X. Biological and Biochemical Properties

XI. Other Aspects

References

4 Isoelectric Focusing on Polyacrylamide Gel

I. Introduction: Isoelectric Focusing in Transition

II. Theory of Isoelectric Focusing

III. Interpretation of pI

IV. Mechanism of Isoelectric Focusing

V. Synthetic Carrier Ampholytes

VI. Apparatus: Analytical and Preparative

VII. Gel

VIII. Protein Fractionation

References

5 Isoelectric Focusing/Electrophoresis in Gels

I. Introduction

II. Aims of Gel Electrofocusing/Gel Electrophoresis

III. General Techniques and Apparatus

IV. Specific Combinations

V. Conclusion

References

6 Isoelectric Focusing in Granulated Gels

I. Introduction

II. Thin-Layer Isoelectric Focusing

III. Preparative Isoelectric Focusing

IV. Applications

V. Technical Appendix

References

7 Continuous-Flow Isoelectric Focusing

I. Introduction

II. Scaling-Up of Electrophoretic Separations

III. Continuous-Flow Electrophoresis

IV. Advantages of Continuous-Flow Isoelectric Focusing

V. Experimental Methods

VI. Practical Considerations

VII. Conclusions

References

8 Isoelectric Focusing in Free Solution

I. Introduction

II. Comparison of Different Methods of Isoelectric Focusing in Free Solution

III. Zone Convection Isoelectric Focusing

IV. Free Zone Electrophoresis

V. Free Zone Isoelectric Focusing

VI. Free Isoelectric Focusing

VII. The Μicroheterogeneity of Proteins

References

9 Transient State Isoelectric Focusing

I. Introduction

II. Instrumental Aspects

III. Data Acquisition and Processing

IV. Methodological Parameters

V. Kinetics of Defocusing and Refocusing

VI. Remarks

References

Index