Two-dimensional gel electrophoresis

Two-dimensional gel electrophoresis (2D-GE) is a form of gel electrophoresis, a laboratory method used to separate mixtures of proteins or other molecules based on their physical properties. 2D-GE is particularly useful for separating proteins that are similar in size but differ in other properties, such as their isoelectric point.

Overview

In two-dimensional gel electrophoresis, proteins are separated in two steps, or dimensions. In the first dimension, proteins are separated based on their isoelectric point, a property that reflects the overall charge of the protein. This is achieved using a technique called isoelectric focusing (IEF). In the second dimension, proteins are separated based on their size using SDS-PAGE, a type of gel electrophoresis.

Procedure

The procedure for two-dimensional gel electrophoresis involves several steps:

1. Sample preparation: The protein sample is prepared by extracting proteins from cells or tissues and then solubilizing them in a suitable buffer.
2. First dimension (IEF): The protein sample is applied to an immobilized pH gradient (IPG) strip, which has a pH gradient along its length. An electric field is applied, causing proteins to migrate along the strip until they reach the pH that matches their isoelectric point.
3. Second dimension (SDS-PAGE): The IPG strip is then placed on top of a polyacrylamide gel, and an electric field is applied. This causes proteins to migrate through the gel based on their size, with smaller proteins moving faster than larger ones.
4. Detection and analysis: After electrophoresis, the proteins in the gel can be detected using various methods, such as staining or Western blotting. The resulting pattern of protein spots can be analyzed using software to identify individual proteins and compare different samples.

Applications

Two-dimensional gel electrophoresis is widely used in proteomics, the study of the entire set of proteins expressed by a genome, cell, tissue, or organism. It can be used to compare protein expression between different samples, such as healthy and diseased tissues, or to identify proteins that interact with a particular drug or other molecule.

See also

• Protein electrophoresis
• Capillary electrophoresis
• Mass spectrometry

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