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Related Concept Videos

SDS-PAGE01:27

SDS-PAGE

Gel electrophoresis is a method that separates biological macromolecules like nucleic acids or proteins by forcing them to pass through a gel matrix under an electric field.
A variation of gel electrophoresis, termed  polyacrylamide gel electrophoresis (PAGE), is commonly used for separating proteins according to their molecular size by passing them through a polyacrylamide gel. Because of the varying charges associated with amino acid side chains, PAGE can be used to separate intact proteins...
Two-dimensional Gel Electrophoresis01:22

Two-dimensional Gel Electrophoresis

Two-dimensional gel electrophoresis is a high-resolution protein separation method first introduced by O' Farrell and Klose in 1975. This method involves protein separation by two dimensions, mass and charge, making it more accurate than one-dimensional gel electrophoresis.
The first dimension separation uses the isoelectric focusing or IEF technique performed on immobilized pH gradient (IPG) strips that separate proteins according to their isoelectric points.
Biological samples, such as  cells...
Ion-Exchange Chromatography01:09

Ion-Exchange Chromatography

Ion-exchange chromatography, or IEC, is a technique for separating ions based on their affinity for the stationary phase. The stationary phase is a cross-linked polymer resin with covalently attached ionic functional groups. The functional groups can be either positively charged (cation exchangers) or negatively charged (anion exchangers). A cation exchanger consists of a polymeric anion and active cations, while an anion exchanger is a polymeric cation with active anions. The choice of...
Electrophoresis: Overview01:20

Electrophoresis: Overview

Electrophoresis is a powerful analytical separation technique that relies on the differential migration of charged species when subjected to an electric field. The core strength of electrophoresis lies in its ability to separate high-molecular-weight species in complex mixtures. It has found widespread use in biochemistry, molecular biology, and analytical chemistry, allowing the separation of compounds like amino acids, nucleotides, carbohydrates, and proteins with excellent resolution.
There...
Capillary Electrophoresis: Instrumentation01:20

Capillary Electrophoresis: Instrumentation

Capillary electrophoresis instrumentation typically consists of several key components. A high-voltage power supply generates the electric field necessary for the separation by connecting to an anode (the positively charged electrode) and a cathode (the negatively charged electrode) located in buffer reservoirs at each end of the capillary tube. The system includes a sample vial, a fused silica capillary tube coated with polyimide for mechanical strength through which the sample components...
Capillary Electrophoresis: Applications01:30

Capillary Electrophoresis: Applications

Capillary electrophoretic separations offer various modes, each with unique applications. These modes include capillary zone electrophoresis, capillary gel electrophoresis, capillary array electrophoresis, capillary isoelectric focusing, capillary isotachophoresis, micellar electrokinetic chromatography, and capillary electrochromatography.
Capillary zone electrophoresis (CZE) separates ionic components based on their electrophoretic mobility. It has been used to separate proteins, amino acids,...

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Related Experiment Video

Updated: Jul 17, 2026

On-chip Isotachophoresis for Separation of Ions and Purification of Nucleic Acids
10:32

On-chip Isotachophoresis for Separation of Ions and Purification of Nucleic Acids

Published on: March 2, 2012

Isoelectric focusing as the crow flies.

P G Righetti1

  • 1Department of Biomedical Sciences and Technologies, University of Milan, Italy.

Journal of Biochemical and Biophysical Methods
|June 1, 1988
PubMed
Summary

Isoelectric focusing has evolved through four generations, from early diffusion methods to current immobilized pH gradients. This advancement offers improved protein separation techniques for scientific research.

Area of Science:

  • Biochemistry
  • Analytical Chemistry
  • Separation Science

Background:

  • Isoelectric focusing (IEF) is a technique for separating proteins based on their isoelectric point (pI).
  • The development of IEF has progressed significantly over several decades.
  • Early methods faced limitations in stability and resolution.

Purpose of the Study:

  • To trace the historical evolution of isoelectric focusing methodologies.
  • To classify and discuss the four major generations of IEF techniques.
  • To highlight the advancements leading to immobilized pH gradients.

Main Methods:

  • Review of historical literature and seminal works in isoelectric focusing.
  • Classification of IEF into four distinct methodological generations.

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Highly Sensitive and Quantitative Detection of Proteins and Their Isoforms by Capillary Isoelectric Focusing Method
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Separation of Bioactive Small Molecules, Peptides from Natural Sources and Proteins from Microbes by Preparative Isoelectric Focusing (IEF) Method
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Separation of Bioactive Small Molecules, Peptides from Natural Sources and Proteins from Microbes by Preparative Isoelectric Focusing (IEF) Method

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Highly Sensitive and Quantitative Detection of Proteins and Their Isoforms by Capillary Isoelectric Focusing Method

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  • Discussion of the principles and characteristics of each generation.

    • Main Results:

    • Generation A: Kolin's two-step approach (diffusion gradient, then separation).
    • Generation B: Svensson-Rilbe's carrier ampholyte approach for pH gradient formation.
    • Generation C: Immobilized pH gradients (IPGs) using grafted non-amphoteric buffers.
    • Generation D: Mixed-bed IPGs combining soluble and immobilized gradients.

    Conclusions:

    • The evolution of IEF demonstrates a progression towards more stable and high-resolution separation techniques.
    • Immobilized pH gradients represent a significant advancement, offering superior reproducibility and versatility.
    • IEF continues to be a vital tool in protein analysis and biochemistry.