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Electrophoresis: Introduction, Principle, Factors Affecting Electrophoretic Mobility, Classification

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:

Here's the diagram illustrating the "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:

Here's the diagram illustrating the "Factors Affecting Electrophoretic Mobility in Electrophoresis"

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:

Here's the diagram illustrating the "Classification of Electrophoresis"

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.

    1. Agarose Gel Electrophoresis: Typically used for the separation of DNA and RNA fragments.

    2. 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.


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