Electrophoresis Lecture Notes

Definition

• Electrophoresis: Movement of charged particles in a liquid medium under an electric field.
• Discovery: First observed by a Russian professor studying the effect of an electric field on dispersed clay particles in water.

Applications

• Used for separation of biomolecules such as DNA, RNA, and proteins.
• Most biomolecules have a charge:
  • DNA/RNA: typically have a net negative charge.
  • Proteins: can have either positive or negative charge depending on amino acids.

Setup and Parameters

• Buffer: Electrophoresis is performed in a buffer with electrodes connected to a power pack.
• Power Pack Readings:
  • Voltage (potential difference across electrodes).
  • Current (flow of electricity across electrodes).

Important Factors

• Distance between Electrodes:
  • Key to calculating electric field (E = V/d).
  • Units: Volts per centimeter.

Example Calculation

• Distance: 10 cm, Voltage: 100 V → Electric field = 10 V/cm.
• Distance: 20 cm, Voltage: 100 V → Electric field = 5 V/cm.
• Time for DNA Agarose Gel Electrophoresis:
  • Shorter electric field → faster completion (15-20 minutes for 10 cm vs. doubled time for 20 cm).

Current and Heat Generation

• Current Measurement: Measured in milliamps.
• Heat Generation:
  • Depends on current, voltage, and time: H = V * I * t.
  • Voltage relationship: V = I * R (Resistance depends on buffer type, ionic strength, and gel used).
  • Heat equation: H = I² * R * t (Heat increases fourfold if current doubles).
  • Advice: Carry out electrophoresis at low current to avoid excessive heat.*

Types of Electrophoresis

1. Paper Electrophoresis:

   • Simple technique using a wet filter paper strip in buffer.
   • Sample molecules separate based on net charge.

2. Agarose Gel Electrophoresis:

   • Widely used for DNA/RNA.
   • Samples loaded in small wells, migrate from negative to positive electrode.
   • Smaller DNA fragments migrate faster.

3. Capillary Electrophoresis:

   • Conducted in a capillary tube immersed in buffer.
   • Sample detection via UV absorbance, IR absorbance, or refractive index change.

4. SDS-PAGE (Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis):

   • Used for protein separation in a vertical unit.

5. Two-Dimensional Electrophoresis:

   • First step: isoelectric focusing in a pH gradient gel.
   • Second step: SDS-PAGE to separate based on molecular weight.

6. Pulsed Field Gel Electrophoresis:

   • For separation of chromosomes or large DNA fragments.
   • Uses four electrodes applying alternating electric fields at 120-degree angles.