Electrophoresis: Separating Charged Molecules

Electrophoresis: Separating Charged Molecules

Electrophoresis is a technique that separates charged particles, such as proteins, nucleic acids, and other biomolecules, based on their size, shape, and charge. This method was first introduced by Tiselius in 1937 and has since become an essential tool in various fields, including biology, chemistry, and medicine.

Principle of Electrophoresis

The principle of electrophoresis is based on the movement of charged particles under an electrical field. Biomolecules, such as proteins and DNA, possess electrical charges due to their chemical composition. When an electric field is applied, these charged particles migrate towards the oppositely charged electrode.

Components of an Electrophoresis Apparatus

An electrophoresis apparatus typically consists of the following components:

1. Buffer: The buffer solution, which carries the current and maintains the pH of the medium, is essential for the proper functioning of the electrophoresis apparatus.
2. Support medium: This is the matrix in which the separation takes place. Examples of support media include agarose, cellulose acetate, and polyacrylamide.
3. Power supply: The power supply provides the electrical field necessary for the movement of charged particles.
4. Cover: The cover reduces evaporation of buffer and prevents contamination during the electrophoretic run.
5. Densitometer: The densitometer is used for quantification of separated bands by comparison of the optical density of bands.

Factors Affecting Electrophoretic Mobility

Several factors influence the mobility of molecules during electrophoresis:

1. Size: The size of the molecule is inversely proportional to its mobility.
2. Charge: The net charge of the molecule is directly proportional to its mobility.
3. Shape: The shape of the molecule can also affect its mobility.
4. Strength of the electrical field: The strength of the electrical field is proportional to the mobility of the molecules.
5. Buffer: The buffer's pH and ionic strength can affect the mobility of molecules.
6. Support medium: The support medium's pore size, gel concentration, and affinity for the molecules in the sample can influence the resolution of the separation.

Types of Support Media

Various support media are used in electrophoresis to separate different molecules effectively:

1. Agarose gel: This is a common medium for DNA separation, particularly for agarose gel electrophoresis.
2. Cellulose acetate membrane: This medium is suitable for proteins and has a lower run-time compared to agarose gel.
3. Polyacrylamide: This medium is often used for protein separation and can be adjusted to separate molecules with different sizes and charges.

Electrophoresis is a powerful technique used to separate and analyze biomolecules, providing valuable insights into the structure, function, and regulation of biological systems.