MEVE 018: Unit 09 - Electrophoresis

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UNIT 9: ELECTROPHORESIS

9.0 Introduction

Electrophoresis is a powerful analytical technique used to separate charged biomolecules such as DNA, RNA, and proteins based on their size, charge, or shape under the influence of an electric field. It plays a significant role in molecular biology, biotechnology, forensic science, and environmental monitoring. This unit explores the principles, types, methodologies, and applications of electrophoresis.

9.1 Objectives

After completing this unit, learners will be able to:

  • Understand the fundamental principles of electrophoresis.
  • Differentiate between various types of electrophoresis.
  • Describe the components and procedures of gel electrophoresis.
  • Understand capillary and 2-D gel electrophoresis.
  • Recognize the application of electrophoresis in environmental science.

9.2 General Principle of Electrophoresis

Electrophoresis is based on the migration of charged molecules in an electric field. When an electric current is applied, molecules move toward the electrode of opposite charge:

  • Cations migrate toward the cathode (negative electrode).
  • Anions migrate toward the anode (positive electrode).

The rate of migration depends on:

  • Net charge of the molecule
  • Molecular size
  • Shape
  • Type of medium (gel, capillary)
  • Voltage applied

Electrophoresis enables separation and analysis of biological molecules, especially useful for diagnostics and monitoring environmental contaminants.

9.3 Types of Electrophoresis

Electrophoresis can be classified based on medium or method:

  1. Free solution electrophoresis (no supporting medium)
  2. Zone electrophoresis (using supporting media like gels)
  3. Paper electrophoresis
  4. Gel electrophoresis
  5. Capillary electrophoresis
  6. 2-Dimensional gel electrophoresis (2-DE)

Each type has specific applications depending on the nature of the analytes and the resolution required.

9.4 Gel Electrophoresis

Gel electrophoresis is one of the most commonly used techniques in molecular biology.

9.4.1 Types of Gels

  • Agarose Gel: Commonly used for DNA and RNA separation; gel concentration determines the resolution.
  • Polyacrylamide Gel (PAGE):
    • Native PAGE: Maintains protein structure.
    • SDS-PAGE: Denatures proteins and separates them based on molecular weight.

9.4.2 Gel Staining

After electrophoresis, the separated molecules must be visualized:

  • Ethidium Bromide (EtBr): Binds to DNA and fluoresces under UV light.
  • SYBR Green/SYBR Safe: Safer alternatives to EtBr.
  • Coomassie Brilliant Blue: Used to stain proteins.
  • Silver Staining: Very sensitive, used for protein detection.

9.4.3 Preparation and Running of Agarose Gels

Steps:

  1. Prepare agarose solution in buffer (e.g., TAE, TBE).
  2. Pour into a casting tray with a comb to form wells.
  3. Allow to solidify.
  4. Load DNA/RNA samples mixed with loading dye into the wells.
  5. Run electrophoresis at constant voltage.
  6. Visualize under UV or blue light.

Gel electrophoresis allows the estimation of molecular weight and purity of samples.

9.5 Capillary Electrophoresis

Capillary electrophoresis (CE) uses narrow capillary tubes filled with buffer, allowing very efficient and high-resolution separation.

Advantages:

  • High speed and efficiency
  • Small sample and reagent volume
  • Automated and computer-controlled

Applications:

  • Environmental water and soil sample analysis
  • Detection of pollutants and contaminants
  • DNA sequencing and mutation analysis

CE is particularly suitable for the analysis of ionic pollutants, pesticides, and heavy metals in environmental monitoring.

9.6 2-D Gel Electrophoresis

2-Dimensional Gel Electrophoresis is a high-resolution technique used to separate complex mixtures of proteins.

Steps:

  1. First Dimension: Isoelectric focusing (IEF) separates proteins based on isoelectric point (pI).
  2. Second Dimension: SDS-PAGE separates based on molecular weight.

Applications:

  • Proteomics
  • Identification of biomarkers
  • Analyzing differential protein expression due to pollutants or stress
  • Comparative studies of microbial communities

9.7 Application of Electrophoresis Techniques in Environmental Monitoring and Analysis

Electrophoresis is crucial in analyzing environmental samples:

  • Detection of pollutants like pesticides, heavy metals, and PCBs (using protein markers).
  • Microbial diversity analysis in soil, water, and wastewater.
  • DNA fingerprinting for tracking environmental bacteria.
  • Monitoring genetically modified organisms (GMOs) in ecosystems.
  • Studying effects of pollution on gene expression in indicator species.

In environmental biotechnology, electrophoresis serves as a diagnostic tool to evaluate ecosystem health and contamination levels.

9.8 Let Us Sum Up

  • Electrophoresis is an essential technique for separating biomolecules based on their size and charge.
  • Types include gel electrophoresis, capillary electrophoresis, and 2-D electrophoresis.
  • Gel types, staining methods, and electrophoretic conditions influence resolution and interpretation.
  • Environmental applications include pollutant detection, microbial monitoring, and biomarker analysis.

Mastery of electrophoresis techniques is critical for environmental scientists, biotechnologists, and researchers in molecular studies.

9.9 Keywords

  • Electrophoresis-Technique to separate charged molecules using an electric field.
  • Gel Electrophoresis-Uses a gel matrix to separate DNA, RNA, or proteins based on size.
  • Agarose Gel-Gel used for nucleic acid separation, made from seaweed extract.
  • Polyacrylamide Gel-Used for protein separation; can be denaturing (SDS) or native.
  • Staining-Process of visualizing separated biomolecules in a gel.
  • Capillary Electrophoresis (CE)-Uses narrow capillaries for high-resolution separation of small analytes.
  • 2-D Gel Electrophoresis-Separates proteins based on charge and molecular weight.
  • Isoelectric Focusing-Separation of proteins based on their isoelectric points.
  • Environmental Monitoring-Use of analytical tools to assess pollutants or contaminants in ecosystems.
  • Loading Dye-Colored solution mixed with samples to track migration in gel electrophoresis.

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