Electrophoresis - Revised (Winter 2025)

Overview of Electrophoresis

  • Electrophoresis: The movement of charged molecules in a solution caused by an electric field.

  • Gel Electrophoresis: Specific technique where charged molecules migrate through a gel under electric current.

Safety Precautions

  • High risk of electric shock with electrophoresis equipment.

  • Precautions:

    • Use electrophoresis equipment with electrical interlocks to interrupt current flow when opened.

    • Regularly verify the integrity of the electrophoresis tank for leaks/damage.

    • Keep apparatus in low traffic areas and use physical barriers to discourage accidental contact.

    • Properly display warnings about high voltage.

Electrophoresis Purpose and Direction

  • Main Purpose:

    • To separate biological molecules such as DNA, RNA, or proteins.

  • Direction: Molecules move towards the positive (+) electrode.

Gels

  • Definition: A gel is a Jello-like matrix with pores that allows biological molecules to migrate through it.

  • Components: Made from acrylamide or agarose.

Agarose Gels

  • Composition: Derived from seaweed, available in powdered form, and is soluble in boiling water.

  • Properties:

    • Solidifies at room temperature.

    • The amount of agarose used determines gel density.

  • Applications:

    • Estimating size of DNA fragments, analyzing PCR products, examining DNA/RNA integrity, purifying fragments, separating fragments prior to blotting, screening proteins for abnormalities.

Polyacrylamide Gels

  • Definition: Comprises a mixture of acrylamide and bis-acrylamide which polymerizes to form a mesh.

  • Types:

    • Non-denaturing: 29:1 ratio used for separating small/closely sized DNA.

    • Denaturing: 19:1 ratio used for separating single-stranded DNA.

  • Applications: High resolution fragment separation, protein screening, SDS-PAGE techniques.

Agarose vs Polyacrylamide Gels

  • Agarose:

    • Easy to prepare, non-toxic, lower resolution, lower voltage (~100V).

  • Polyacrylamide:

    • Higher resolution, can load larger quantities, higher voltage (~300V), neurotoxin requiring careful handling.

Gel Electrophoresis Power

  • Mechanism:

    • Molecules move in response to an applied electric field.

    • The relationship of Voltage (V), Current (I), and Resistance (R) follows Ohm's law: I = V/R.

  • Power Equation: P = V x I (Power increases with heat generated during electrophoresis).

Approaches to Power Control

  • Constant Current:

    • Maintains current as resistance increases, leading to increased heat.

    • Shorter run times, sharper bands but harder with multiple gels.

  • Constant Voltage:

    • Keeps voltage steady while current decreases with resistance, reducing heat and allowing multiple gels but longer run times.

  • Constant Power:

    • Maintains power despite resistance increase but results in unpredictable migration rates.

Factors Affecting Migration

  • Molecular Size and Shape:

    • Migration speed affected by size, conformation, and gel concentration.

  • Ionic Strength of Buffer:

    • Essential for maintaining electric current flow; affects the mobility of molecules.

  • Sample Conditions:

    • Inappropriate pH and high salt concentrations can distort bands and affect results.

Visualization Methods

  • DNA/RNA Stains:

    • Ethidium Bromide: Fluorescent, mutagenic.

    • SYBR Green/Safe: Safer alternatives, more sensitive.

  • Protein Stains:

    • Coomassie: Common dye for in-gel protein staining, works quickly and with minimal reagents.

Specific Techniques

  • Types of Gel Electrophoresis:

    • Serum Protein Electrophoresis: Differentiates plasma proteins; diagnosed conditions include cancer and liver issues.

    • SDS-PAGE: Denatures proteins for size sorting; uses heat and reducing agents.

  • Immunoelectrophoresis: Combines antigen separation and antibody precipitation for identification.

  • Capillary Electrophoresis: High-resolution technique allowing the separation of small volumes with high throughput.

Applications of Electrophoresis

  • Used for molecular analysis in genetics, proteomics, and diagnostics, including applications like sequencing, fragment analysis, and more.