Two-Dimensional Gel Electrophoresis: Overview & Applications

Two-Dimensional Gel Electrophoresis: Overview & Applications

Two-Dimensional Gel Electrophoresis: Overview

Gel electrophoresis is an essential lab technique for separating DNA, RNA, and proteins. The technique has been modified in many ways to serve different purposes, such as agarose gel electrophoresis, polyacrylamide gel electrophoresis, and starch electrophoresis.  

One such modified electrophoretic method is two-dimensional gel electrophoresis, also known as 2-DE, 2-D electrophoresis, or 2D-PAGE. The technique was first independently introduced by two scientists named O'Farrell and Klose in 1975 for the detection, quantification, and analysis of proteins. 

In this methodology, a mixture of protein samples is separated based on two different properties of proteins in two dimensions. 

  • In the second dimension, proteins are separated based on their sizes or molecular weight through sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).   

2D-gel electrophoresis is widely used in biochemistry and proteomics research studies to detect and analyze many known and unknown proteins, such as membrane proteins, ribosomal proteins, or proteins of other organisms.                                                                

Figure: The summary of a 2-D gel electrophoresis. 

In this article, we will review the working mechanism of 2-D gel electrophoresis and its applications, and learn about industries that frequently employ the technique in their research workflows. 

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How Does Two-Dimensional Gel Electrophoresis Work?

2-dimensional gel electrophoresis assay consists of two separation steps:

  • First Dimension: In the first dimension, the technique separates protein molecules based on their isoelectric point (pI). Various methodologies, such as immobilized gradient electrophoresis (IPEG), isoelectric focusing (IEF), or non-equilibrium pH gradient electrophoresis (NEPHGE), are used to recover strong protein bands by subjecting them to an immobilized pH gradient created by IPG strips. The step separates proteins within their specific pH range. 

Figure: Separation of proteins based on charges on an IPG strip in the presence of an electric field. 

Before this step, the protein sample is prepared in a concentration and solution required for the workflow. The method of sample preparation is based on solubility and native charge of the protein of interest, removing impurities, such as abundant proteins, nucleic acids, salts, and nucleic acids. 

  • Second Dimension: In the second dimension, we perform protein separation based on molecular weight using SDS Laemmli or Tris-Tricine buffers.

Performing isoelectric focusing (IEF) in scientific research introduces various complexities, such as:

  • Proteins tend to exhibit decreased solubility and may even undergo precipitation as they approach their isoelectric point (pI), especially in low-salt buffers that are designed for IEF. 
  • The presence of IEF gels and buffers can interfere with sample preparation for mass spectrometry (MS), making staining and subsequent analysis challenging. 

Consequently, IEF is typically conducted as the initial step to render the sample compatible with MS analysis by SDS-PAGE.

Figure: The separation of proteins based on their sizes in an acrylamide gel. 

To enhance reproducibility and accuracy, various techniques and reagents are utilized. These include the addition of reducing agents such as dithiothreitol (DTT) to prevent oxidation and the use of specific dyes or labeled antibodies for targeted protein detection.

The next step after the second dimension is the visualization of the target protein through fluorescent protein tags or total protein stains. This is followed by image acquisition and analysis and protein spot excision, digestion, and identification of obtained peptides. 

Additional steps like solubilization, chromatography, and protein fractionation may be employed for further purification and analysis.

Figure: the diagram of the process of 2-D gel electrophoresis.

What is Two-Dimensional Gel Electrophoresis Used For?

2-DE is a widely used technique in molecular biology and proteomics studies for the detection and analysis of proteins of interest from a complex mixture. Additionally, it has various applications in whole proteome analysis, detection of biomarkers and disease markers, cancer research, cell differentiation, bacterial pathogenesis, drug discovery, microscale protein purification, and product characterization. 

Protein Detection

2D gel electrophoresis has extensive application for detecting alterations in protein expression levels caused by diseases or drug interventions. It serves as a valuable tool for studying post-translational modifications like oxidation or phosphorylation and identifying protein isoforms. Even small variations in protein mass or isoelectric point (pI) result in shifts in protein patterns, observed through 2-D gel electrophoresis.

Protein Analysis

2D gel electrophoresis (2-DE) is a powerful methodology to separate thousands of proteins at once, which makes it an exceptional method for analyzing complex protein mixtures. The technique enables researchers to visually confirm any alterations in protein and post-translational modifications (PTMs) abundance, providing early validation for downstream analytical steps. 

2-DE detects post- and co-translational modifications that cannot be even predicted from the genomic sequence.

What Industries Use Two-Dimensional Gel Electrophoresis?

2-D electrophoresis is a gel-based technique widely utilized in proteomics studies.

It involves several key steps and components, including:

  • Solubilization of protein extracts using urea and detergents
  • Fractionation of proteins based on their isoelectric point (pI) using immobilized pH gradient (IPG) strips and ampholytes
  • Separation based on molecular weight using acrylamide gels
  • Coomassie or silver staining for visualizing protein bands
  • Gel analysis using imaging techniques such as image analysis software.


In biotech research, 2-D gel electrophoresis is extensively used for the quantification and analysis of proteins. It’s a powerful technique for studying protein composition, modifications, and interactions. It offers high resolution, reproducibility, and the ability to analyze complex protein extracts. The combination of gel-based separation, IPG strips, labeling methods, and image analysis software enables comprehensive protein profiling and investigation in various research fields.

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Procure Your R&D Lab Needs with Excedr

Two-dimensional electrophoresis (2DE) is a traditional technique used to separate proteins based on their size (sodium dodecyl sulfate-polyacrylamide gel electrophoresis, SDS-PAGE) and their charge (isoelectric focusing, IEF) in the presence of an electric field. 

The technique is widely used in proteome analysis and profiling of proteins from a mixture. The obtained results help in the understanding of the functioning of the organisms and diseases treatments. 

Given the significant use in medical studies that can affect human life, the use of high-throughput workflows necessitates advanced instruments and high-quality reagents to ensure the acquisition of accurate and reliable data. However, it increases the overall cost of the assay and makes it challenging to run the lab. 

This is made easier through Excedr’s effective and efficient leasing solution

The program enables biotech researchers and founders to acquire their desired equipment or outfit their whole lab on lease. Our team can work closely with you to create a lease agreement that allows you to acquire the equipment you need without overspending your operating budget.

We have a range of equipment available at Excedr, including analytical equipment, biotech and life sciences equipment, and clinical equipment. The leasing program covers both the upfront cost and the costs associated with the repair and maintenance of equipment. Thus, allowing you to spend your budget on other lab operations. 

With Excedr's leasing program, you gain the advantage of saving money and time while enjoying enhanced operational and financial flexibility in your lab. This empowers you to expedite your research and development activities, giving you a competitive edge in your field.