Electrophoresis

Introduction

• Discovery: In 1937, Arne Tiselius demonstrated separation of charged particles using an electrical field.
• Biomolecules: Proteins, peptides, nucleic acids, and nucleotides migrate based on net charge.
• Medium: Transitioned from liquid to solid support media (e.g., Whatman filter paper, agarose).

Components of Electrophoresis Apparatus

• Buffer: Carries current, maintains pH.
• Wicks: Connect support medium with buffer.
• Support Medium: Matrix for separation.
• Cover: Reduces evaporation and contamination.
• Power Supply: Provides electrical field.
• Densitometer: Quantifies separated bands.

Factors Affecting Electrophoretic Mobility

• Size, Shape, and Net Charge:
  • Mobility inversely proportional to size; directly proportional to charge.
  • Anions move to anode (+), cations to cathode (-).
• Electrical Field Strength:
  • Mobility proportional to voltage, inversely to resistance.
• Buffer:
  • Ionic strength affects migration speed and heat generation.
  • pH affects ionization and migration direction.
• Supporting Medium:
  • Pore size and affinity affect migration; electroendosmosis can reduce resolution.

Types of Support Medium

• Whatman Filter Paper: Long runtime, low voltage, poor resolution.
• Cellulose Acetate: Short runtime, high resolution, expensive.
• Agarose Gel: Separates proteins, nucleic acids; affected by electroendosmosis.
• Polyacrylamide Gel: Excellent resolution; precise pore size control.

Variants of Electrophoresis

• Isoelectric Focusing: Separates by isoelectric pH; high resolution.
• Immunoelectrophoresis: Antigen-antibody reactions identify specific proteins.
• High-voltage Electrophoresis: Fast separation with high voltage.
• Pulsed-field Electrophoresis: Separates long nucleotide fragments.
• Capillary Electrophoresis: High voltage, speedy separation, and quantification.
• Two-dimensional Electrophoresis: Combines isoelectric focusing and SDS-PAGE.

Specimen Requirements

• Biological Specimens: Serum, plasma, whole blood, nucleic acid extracts for diagnostics and research.

Testing Procedures

• Sample Processing: Centrifugation, preparation of hemolysate, use of PCR products.
• Separation and Quantification: Use of densitometry after running the electrophoresis.

Interfering Factors

• Heat: Increases molecule motion, reducing resolution.
• Adsorptive Groups: Bind analytes, hindering mobility.
• Electroendosmosis: Opposes analyte motion, reducing resolution.

Results and Reporting

• Staining and Visualization: Staining indicates presence, densitometry quantifies bands.
• Clinical Insight: Abnormal patterns can indicate diseases.

Clinical Significance

• Diagnosis of Hemoglobinopathies and Thalassemia: Abnormal hemoglobin patterns guide diagnosis.
• Utility in Research: Used in genomics, proteomics, and forensic science.

Quality Control and Lab Safety

• Controls and Precautions: Use controls, avoid carcinogenic materials, and ensure safety in preparation and visualization.

Enhancing Healthcare Team Outcomes

• Interprofessional Team: Collaboration among specialists enhances diagnostic accuracy and patient care.