Isoelectric Focusing Electrophoresis: Overview & Application

Isoelectric Focusing Electrophoresis: Overview & Application

Isoelectric Focusing Electrophoresis: Overview

Isoelectric focusing (IEF) is a molecular technique used in labs to separate different molecules based on the difference in their pI or isoelectric point. The technique is also known as electrofocusing.

Basically, it is a type of zone electrophoresis that uses the pH of the gel to determine the overall charge on the molecule of interest. In the first step, the proteins are separated by their pI value and then SDS-PAGE is used to separate them further based on their molecular weight.

Figure: An illustration of the resolution of proteins on pH 3–10 IPG strips based on their pI values.

In this technique, the separation of proteins is done by applying an electric field within the pH gradient. As the molecules move through the gradient in response to the voltage, they start to separate based on their size.

Upon reaching a pH value that matches its pI, a protein’s net charge becomes neutral, and it stops migrating. Thus, each protein in the sample is focused according to its pI.

IEF or Isoelectric focusing can be performed by using:

  • Carrier ampholytes that migrate through the gel to generate pH gradients.
  • Immobilized pH gradients (IPG) with ampholytes covalently bound to an acrylamide gel

Isoelectric focusing (IEF) is widely used in molecular biology labs and biotech labs because it has better resolution and quantitation than the gel electrophoresis technique. Further, it’s easier to perform as there’s no stress of the placement of samples.

Today, many types of IEF have been developed, some of which include:

  • Microfluidic chip-based IEF: Electrophoresis using microchips offers a number of advantages over capillary electrophoresis, including:
  • Rapid protein analysis
  • Easy integration with other microfluidic units
  • Whole channel detection
  • Nitrocellulose films
  • Smaller sample sizes
  • Lower fabrication costs.
  • Capillary isoelectric focusing: A high-resolution method of separating molecules based on their pI point in fused-silica capillaries with internal diameters of 25-75 μM. The workflow can be easily automated in labs.
  • IEF-gel electrophoresis: A widely used technique in isoelectric focusing (IEF). It effectively minimizes convection and incorporates a gel-sieving effect to achieve precise separation of proteins according to their size.

In this article, we will cover more about isoelectric focusing, including its working mechanism, its applications, and the industries that frequently use techniques.

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How Does Isoelectric Focusing Electrophoresis Work?

Isoelectric focusing is a technique that works by applying an electric field to the pH gradient medium containing protein samples.

Initially, proteins start moving towards their electrode with the opposite charge. However, as soon as they start migrating through the pH gradient gel matrix, they either lose or pick up protons, which results in a decrease in their electrophoretic mobility and net charge. Thus, the proteins start slowing down and eventually stop after arriving at the isoelectric point.

If proteins diffuse in a region with pH values lower than its pI, they become protonated and move toward the cathode in the presence of the electric field. Whereas, if proteins move to a region with greater pH values, they become negatively charged and move toward the anode.  In this way, the separated proteins are focused in a sharp band in the pH gradient based on their respective pI values.

In the pH gradient medium, proteins move at different rates toward their pI values. However, once reached, they stick to those pH values for an extended time. Further, in IEF proteins reach their steady-state positions from anywhere in the gel.

To separate focused proteins based on their molecular weight, the gel is incubated in an SDS buffer and applied on SDS–PAGE slab gels. In addition to micro-range IPG strips (of different lengths and pH ranges), electrophoretic cells are used to achieve the highest resolution in IEF results.

The relative focusing power of two strips with the same length but different pH ranges can be determined by comparing the ratio of their pH ranges in terms of the number of pH units

What is Isoelectric Focusing Electrophoresis Used For?

Isoelectric-focusing electrophoresis plays a crucial role in a variety of life sciences fields, spanning protein analysis, purification, proteomics research, and clinical diagnostics. It provides valuable insights into the separation and characterization of biomolecules based on their charge.

Molecule Separation

IEF is used to separate proteins in a mixture (mostly peptides, proteins, and different amino acid sequences)  based on their pI values in a pH gradient gel matrix in the presence of an electric field. It’s a major procedure involved in the first dimension of a 2-D electrophoresis experiment. The second dimension of protein separation is accomplished by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).

The preparative IEF method is used to isolate proteins from contaminants with slightly different pIs at the end of the purification process. It is usually used to isolate isoforms of proteins. Even small charge differences in the amino acid residues of proteins can be detected by using IEF.

IEF in analytical applications serves multiple purposes, including the evaluation of protein extract complexity and the identification of specific components. However, its exceptional resolving power makes it particularly well-suited for detecting microheterogeneity within purified proteins.

A combination of IEF on agarose gels and immunoblotting is now accepted as the gold standard for detecting oligoclonal Igs.

Quality Control Testing

The application of isoelectric focusing extends to the quality control testing of therapeutic biological products, where it plays a vital role in demonstrating batch consistency. With its high resolving power and sensitivity to detect even subtle changes in protein charge, isoelectric focusing proves to be particularly valuable in this context.

What Industries Use Isoelectric Focusing Electrophoresis?

Isoelectric focusing is an invaluable tool in life sciences and other fields for the separation of molecules in a mixture.


IEF is extensively used in the study and diagnosis of many genetically transmitted disorders by identifying abnormal proteins associated with the disorder.

Further, IEF is frequently employed by immunochemists to analyze a wide range of antigens and preparations. When combined with immunoblotting, this technique proves highly valuable in determining the specific antigen-binding profile of an antiserum or monoclonal antibody, enabling precise characterization.


IEF is widely used in soil science to identify matrices in organic fertilizers and assess organic matter stabilization in soil and composts. It provides valuable insights into soil health and nutrient management.

Further, IEF has profound applications in protein and enzyme analysis of oilseeds, cereals, plants, and vegetables. Its utilization in genetic studies has played a crucial role in enhancing the nutritional value of these seeds, driving advancements in agricultural and food science.

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Isoelectric focusing is a technique used in life sciences labs for the separation and analysis of proteins and peptides in protein samples. It works based on the principle of gel electrophoresis. However, the proteins are separated in the gels based on their pI values in a pH gradient medium.

The technique is especially useful to separate proteins of the same molecular weight. After separation, proteins are collected for further downstream applications, such as protein identification, characterization, or purification.

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