Electrophoresis is a separation technique where charged molecules are separated based on their size and charge as they move through a medium under the influence of an electric field.
How Electrophoresis Works: A Step-by-Step Breakdown
The mechanism of electrophoresis hinges on the interplay between electrical forces and the physical properties of the molecules being separated. Here's a detailed look:
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Sample Preparation: The sample, containing a mixture of charged molecules (e.g., DNA, RNA, proteins), is prepared in a buffer solution. The buffer maintains a stable pH, ensuring that the molecules retain their charge.
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Gel Preparation (if applicable): Often, a gel matrix (like agarose or polyacrylamide) is prepared. This gel acts as a sieving medium, influencing the migration of molecules based on their size. The gel provides frictional resistance.
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Loading the Sample: The sample is carefully loaded into wells in the gel or onto a solid support (for capillary electrophoresis).
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Applying the Electric Field: An electric field is applied across the electrophoresis medium. This is done by connecting electrodes to a power supply, creating a positive (anode) and a negative (cathode) terminal.
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Migration: Charged molecules experience a force due to the electric field.
- Negatively charged molecules (anions) are attracted towards the anode (positive electrode).
- Positively charged molecules (cations) are attracted towards the cathode (negative electrode).
- Neutral molecules do not migrate.
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Separation: The rate of migration depends on several factors:
- Charge of the molecule: Higher charged molecules experience a greater force and migrate faster.
- Size and shape of the molecule: Smaller molecules experience less frictional resistance and migrate faster, especially in a gel matrix. Larger or more complex shapes may encounter more resistance.
- Strength of the electric field: A stronger electric field results in faster migration.
- Viscosity of the medium: A more viscous medium slows down migration.
- Pore size of the gel (if applicable): Smaller pore sizes provide greater resistance to larger molecules, enhancing separation based on size.
- Buffer composition and pH: These factors influence the charge of the molecules and the conductivity of the medium.
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Visualization: After electrophoresis, the separated molecules are visualized. This can be done using various staining techniques (e.g., ethidium bromide for DNA, Coomassie blue for proteins) or by using fluorescent labels.
Factors Affecting Electrophoretic Mobility
| Factor | Effect on Mobility |
|---|---|
| Charge of the molecule | Higher charge leads to increased mobility (attraction to opposite electrode) |
| Size of the molecule | Smaller size generally leads to increased mobility (less resistance) |
| Electric field strength | Higher field strength leads to increased mobility |
| Medium viscosity | Higher viscosity leads to decreased mobility |
| Gel pore size | Smaller pore size hinders the movement of larger molecules, decreasing mobility |
Types of Electrophoresis
Electrophoresis encompasses various techniques, each tailored to specific separation needs. Common types include:
- Agarose Gel Electrophoresis: Commonly used for separating DNA and RNA fragments.
- Polyacrylamide Gel Electrophoresis (PAGE): Used for separating proteins and small DNA/RNA fragments.
- Capillary Electrophoresis: Performed in narrow capillaries, offering high resolution and speed.
- SDS-PAGE: A type of PAGE used to separate proteins based on size, after they are denatured and coated with a detergent (SDS) to give them a uniform negative charge.
- Isoelectric Focusing (IEF): Separates proteins based on their isoelectric point (pI).
In summary, electrophoresis separates molecules based on their charge and size by applying an electric field across a medium. The molecules move towards the electrode with the opposite charge, and their speed is influenced by their charge, size, the strength of the electric field, and the properties of the medium.
