Electrophoresis

Electrophoresis is a laboratory technique that separates and analyses charged particles, such as proteins, nucleic acids (DNA, RNA), and other biomolecules, based on their size and charge. The principle of electrophoresis relies on the movement of charged particles in an electric field, allowing for their separation and subsequent analysis.

Basic Principles:

1. Electric Field:

   • When a voltage is applied across a medium (usually a gel or a membrane), charged particles move toward the electrode with the opposite charge. Cations move towards the cathode (negative electrode), and anions move towards the anode (positive electrode).

2. Medium:

   • The separation medium is typically a gel (e.g., agarose or polyacrylamide) or a paper matrix. The medium acts as a sieve, allowing particles to migrate at different rates based on size, charge, and shape.

3. Separation Mechanism:

   • Size: Smaller particles move faster through the medium, while larger particles encounter more resistance and move slower.
   • Charge: Particles with greater or higher mobility move faster toward the electrode of the opposite charge.
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   • Shape: The shape of the particles can also affect their migration rate.

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Types of Electrophoresis:

1. Agarose Gel Electrophoresis:

   • Medium: Agarose gel, which is a polysaccharide derived from seaweed.
   • Applications: Commonly used for separating nucleic acids (DNA, RNA) based on size. It is widely used in molecular biology for DNA fragment analysis, PCR product verification, and DNA sequencing.
   • Procedure: DNA samples are loaded into wells in an agarose gel, and an electric field is applied. DNA fragments migrate through the gel and are visualized using staining agents like ethidium bromide or SYBR Green.

2. Polyacrylamide Gel Electrophoresis (PAGE):

   • Medium: Polyacrylamide gel provides a higher resolution than agarose gel.
   • Types:
     • SDS-PAGE: Separates proteins based on their size after denatured and coated with sodium dodecyl sulfate (SDS), which imparts a negative charge to the proteins.
     • Native PAGE: Separates proteins based on size, charge, and shape while maintaining their native conformation.
   • Applications: Used for protein analysis, including protein purification, molecular weight determination, and protein-protein interaction studies.

3. Capillary Electrophoresis (CE):

   • Medium: A narrow capillary tube filled with a buffer solution.
   • Applications: High-resolution separation of small molecules, peptides, proteins, and nucleic acids. It is used in clinical diagnostics, forensic analysis, and genomics.
   • Procedure: A sample is introduced into a capillary tube, and an electric field is applied. Components are separated based on their charge-to-size ratio and are detected as they pass through a detection window.

4. Isoelectric Focusing (IEF):

   • Medium: A gel with a pH gradient.
   • Applications: Separates proteins based on their isoelectric point (pI), the pH at which a protein has no net charge. It is used for protein characterization and analysis of protein isoforms.
   • Procedure: Proteins migrate in a pH gradient until they reach the point where their net charge is zero.

5. Two-Dimensional Electrophoresis (2D-E):

   • Technique: Combines IEF and SDS-PAGE.
   • Applications: Provides a high-resolution separation of proteins based on their isoelectric point and molecular weight. It is used for complex protein analysis, including proteomics.
   • Procedure: Proteins are first separated by IEF in one dimension and then by SDS-PAGE in the second dimension.

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Applications of Electrophoresis:

1. Molecular Biology:

   • DNA Fragment Analysis: Separates DNA fragments based on size for applications such as restriction fragment length polymorphism (RFLP) analysis, DNA fingerprinting, and sequencing.
   • RNA Analysis: Separates RNA species for gene expression studies and RNA integrity assessment.

2. Protein Analysis:

   • Protein Purification: Separates proteins from complex mixtures for further analysis or use in research.
   • Protein Identification: Identifies proteins based on molecular weight and charge, often combined with mass spectrometry for detailed analysis.

3. Clinical Diagnostics:

   • Haemoglobin Electrophoresis: Identifies abnormal haemoglobin variants in conditions like sickle cell disease and thalassemia.
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   • Protein Electrophoresis: Analyzes protein levels and types in blood samples to diagnose and monitor diseases such as multiple myeloma and liver disorders.

4. Forensic Analysis:

   • DNA Profiling: Uses electrophoresis to separate and analyze DNA samples for identification and comparison in criminal investigations.

5. Biotechnology:

   • Genotyping: Identifies genetic variations and mutations in research and clinical settings.
   • Pharmaceutical Development: Analyzes the purity and structure of drugs and biologics.

Procedure:

1. Preparation:
   • Prepare the electrophoresis medium (gel or capillary) and buffer solution.
   • Prepare and load the sample onto the medium.
2. Running the Electrophoresis:
   • Apply an electric field across the medium.
   • Allow the sample components to migrate based on their size and charge.
3. Detection and Analysis:
   • Visualize the separated components using staining agents or other detection methods.
   • Analyze the results based on the migration patterns and compare them with standards or markers.