What Is the Principle of Separating Proteins by Gel Chromatography

Gel chromatography (also referred to as gel permeation chromatography, gel filtration chromatography, or molecular sieve chromatography) is a widely used technique for protein separation and purification. Its principle is based on differences in the molecular size and shape of proteins, which govern their interaction with the gel medium.

 

The gel medium utilized in gel chromatography is typically composed of materials such as polyacrylamide or agarose, which form a three-dimensional network structure. The pore size of the gel medium influences the diffusion rate of protein molecules, thereby determining the separation efficiency. Protein separation based on molecular size can be achieved by adjusting the pore size of the gel medium. Larger protein molecules are excluded from smaller pores and thus migrate more rapidly through the gel, whereas smaller protein molecules enter the pores and are retained for a longer duration. As a result, proteins of different molecular sizes can be effectively separated.

 

Beyond molecular size, gel chromatography also facilitates separation based on protein shape. Due to the structural diversity of proteins, molecules of different conformations diffuse at varying rates within the gel. Generally, larger, globular proteins diffuse more slowly, whereas smaller, elongated proteins exhibit faster diffusion. Thus, gel chromatography enables shape-based separation by modifying the pore size and properties of the gel medium.

 

Additionally, gel chromatography can employ affinity-based mechanisms for selective protein separation. Affinity chromatography, a specialized form of gel chromatography, involves immobilizing specific ligands or antibodies onto the gel matrix to selectively capture target proteins. This method is widely applied in the purification of specific proteins and the enrichment of protein complexes.

 

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