Electrophoresis Principles and Applications

Study Notes

Electrophoresis: Principles, Types, and Uses

Electrophoresis is a technique separating charged particles (ions) through a solution under an electric field.
The charged particles migrate towards the electrode with the opposite charge (negatively charged particles towards the positive terminal (anode), and positively charged particles towards the negative terminal (cathode)).
Electrophoresis is a technique that uses an electric field to separate molecules based on their size and charge.
The technique has been used since 1807, initially using paper electrophoresis.
Modern electrophoresis systems are automated and offer improved resolution and faster results.

Molecular Charge and Movement

Biological molecules like amino acids, peptides, proteins, nucleic acids, and nucleotides can be positively or negatively charged depending on the pH of the solution.
Nucleic acids, with negatively charged phosphate backbones, move towards the anode during DNA electrophoresis.
Proteins, having both positive and negative charges, can migrate towards the anode or cathode depending on the pH of the solution.
The charge of the molecule affects its movement through the electrophoresis medium.

Factors Affecting Ion Mobility

Inherent factors:
- Charge density
- Molecular weight
- Net charge of the molecule
- Size and shape of the molecule
External factors:
- Electrical parameters (current, voltage, and power)
- Viscosity and pore size of supporting medium
- Temperature
- pH of the buffer

Electrophoresis Instrument

The essential components of an electrophoresis system are a power pack and an electrophoresis unit.
The power pack provides the electric field for migration, and the electrophoresis unit contains the electrodes, electrolyte, and support medium.
The support medium, like agarose gel or polyacrylamide gel, acts as a sieve, separating molecules based on their size and charge.

Procedure of Electrophoresis

Separation:
- Gels are prepared and cast.
- The gels are placed in the electrophoresis chamber.
- The sample is loaded into the gel.
- The electric current is applied, and the molecules migrate through the gel.
Detection:
- Stained with a dye.
- Detected using autoradiography (for radioactive samples).
Quantification:
- Quantified using densitometry or other methods.

Electrophoresis types

Agarose gel electrophoresis: Separates DNA or RNA molecules based on their size.
Polyacrylamide gel electrophoresis (PAGE): Separates proteins based on their size and charge.
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE): Separates proteins based on size only.
Capillary electrophoresis: Separates molecules in a capillary tube filled with buffer.
Isoelectric focusing (IEF): Separates proteins based on their isoelectric point.

Uses of electrophoresis

DNA analysis:
- Identifying genetic mutations.
- Diagnosing genetic diseases.
- Paternity testing.
Protein analysis:
- Identifying protein purity.
- Studying protein interactions.
- Diagnosing diseases.
Clinical and research applications:
- Forensic analysis.
- Drug discovery.
- Biotechnology research.

Description

This quiz covers the fundamentals of electrophoresis, detailing its principles, various types, and applications in biological sciences. Understand how charged particles behave in an electric field and the significance of molecular charge in this technique. Perfect for anyone studying biochemistry or molecular biology.