How Electrophoresis Separates and Purifies Proteins Based on Their Physicochemical Properties
Electrophoresis is a technique that separates and purifies proteins and other biomolecules by exploiting their migration in an electric field. The principle behind this technique is based on several key physicochemical properties of proteins:
1. Protein Charge Characteristics
Proteins are composed of amino acid residues, each of which typically carries a charge. Some amino acids have a positive charge, while others possess a negative charge. At a specific pH, a protein will acquire a net overall charge, which is determined by the sum of the charges of all its constituent amino acids. During electrophoresis, the proteins will migrate toward either the anode or cathode in the electric field depending on the sign of their net charge.
2. Protein Size and Shape
The migration rate of proteins in the electric field is also influenced by their size and shape. Larger proteins or those with complex structures tend to move more slowly because they face greater resistance as they pass through the gel matrix.
3. Function of the Gel
The gel matrix, commonly made of polyacrylamide, plays a critical role in electrophoresis by providing a sieving effect. Smaller protein molecules can pass through the gel's pores more easily, whereas larger molecules face greater hindrance. Consequently, the size of the protein directly impacts its migration rate within the gel.
4. Protein’s Isoelectric Point
Two-dimensional electrophoresis combines polyacrylamide gel electrophoresis and isoelectric focusing to separate proteins based on both their isoelectric point (pI) and molecular mass. The isoelectric point is the pH at which a protein’s net charge is zero, meaning it will not migrate in the electric field at this pH. By using these two methods together, proteins can be separated in one dimension based on size, and in a second dimension based on their isoelectric point.
In summary, electrophoresis separates and purifies proteins by exploiting differences in their physicochemical properties, such as charge, size, shape, and isoelectric point. By carefully selecting and controlling the experimental conditions, electrophoresis can effectively achieve this separation and purification.
MtoZ Biolabs, an integrated chromatography and mass spectrometry (MS) services provider.
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