Introduction:
Electrophoresis is a laboratory technique used in the separation of charged molecules, especially proteins and nucleic acids, based on their size and charge.
When subjected to an electric field, these charged molecules move at different rates towards either the anode (positive electrode) or cathode (negative electrode), allowing for their separation.
Principle of Electrophoresis:
Electrophoresis operates on the principle that charged molecules will move in an electric field.
When a mixture of charged molecules is placed in this field, each type of molecule will migrate at a distinct speed based on its size and charge.
Typically, smaller or more highly charged molecules move more quickly through a medium (like a gel or liquid), while larger or less charged molecules move more slowly.
The differential movement in the electric field allows for the separation of the molecules in the mixture.
Factors Affecting Electrophoretic Mobility:
1. Size and Shape of Molecule:
Larger molecules move slower than smaller ones because they face more resistance in the medium.
2. Charge on Molecule:
The movement is directly proportional to the charge; higher charged molecules move faster.
3. Strength of Electric Field:
Increasing the voltage increases the speed of migration, but too high a voltage can cause overheating.
4. Properties of the Medium:
The type and concentration of the gel or medium can affect the separation.
A denser medium provides more resistance.
5. Buffer pH:
pH can influence the ionization state of the molecule, which in turn affects its charge and movement.
6. Temperature:
Movement usually increases with temperature because molecules gain kinetic energy, but too high temperatures can distort results.
Classification:
1. Paper Electrophoresis:
This uses a paper strip as the supporting medium.
It's mainly used for the separation of small, charged molecules.
2. Gel Electrophoresis:
This is the most common type, where a gel made from agarose or polyacrylamide acts as the matrix.
It provides better resolution compared to paper electrophoresis and is widely used for DNA, RNA, and protein separation.
Agarose Gel Electrophoresis: Typically used for the separation of DNA and RNA fragments.
Polyacrylamide Gel Electrophoresis (PAGE): Suited for protein separation because of its fine pores.
3. Capillary Electrophoresis:
Here, separation takes place in small capillary tubes, which allow for high efficiency and resolution.
It has gained popularity for its quick analysis times and ability to handle minute sample volumes.