Principle:
Electrophoresis is the migration of charged particles (ions, proteins, nucleic acids, etc.) in an electric field through a supporting medium (gel, paper, capillary, etc.) under the influence of an applied voltage. Separation occurs mainly due to differences in charge-to-mass ratio and molecular size/shape.
Electrophoresis is the migration of charged particles (ions, proteins, nucleic acids, etc.) in an electric field through a supporting medium (gel, paper, capillary, etc.) under the influence of an applied voltage. Separation occurs mainly due to differences in charge-to-mass ratio and molecular size/shape.
1. Fundamental Driving Forces
Two main opposing forces act on the charged molecule:
| Force | Direction | Responsible for | Mathematical expression |
|---|---|---|---|
| Electrostatic force (driving) | Toward oppositely charged electrode | Movement | Fe = qE |
| Frictional (drag) force (opposing) | Opposite to direction of motion | Limiting velocity | Ff = fv (Stokes' law approximation) |
At terminal velocity: qE = fv → $v = \frac{Eq}{f}$
where $v$ = migration velocity, $q$ = net charge, $f$ = frictional coefficient, $E$ = electric field strength
2. Factors Affecting Electrophoretic Mobility (μ)
Electrophoretic mobility $μ = \frac{v}{E}$ = $\frac{q}{f}$
Mobility depends on:
Mobility depends on:
- Net charge (q) – pH dependent (especially for proteins)
- Size & shape – larger or more asymmetric molecules experience greater friction
- Viscosity of medium
- Ionic strength & temperature
3. Main Types of Electrophoresis (Comparison)
| Type | Medium | Separation mainly based on | Typical analytes | Common application |
|---|---|---|---|---|
| Agarose gel electrophoresis | Agarose (0.5–3%) | Size (molecular sieving) | DNA, large RNA | DNA fragment analysis, PCR products |
| Polyacrylamide gel electrophoresis (PAGE) | Polyacrylamide (3–30%) | Size (very high resolution) | Proteins, small DNA/RNA | Protein purity, SDS-PAGE |
| SDS-PAGE | Polyacrylamide + SDS | Molecular weight (charge masked) | Proteins | Protein MW determination |
| Native PAGE | Polyacrylamide (no SDS) | Charge + size + shape | Native proteins, enzymes | Activity staining, complexes |
| Isoelectric focusing (IEF) | Polyacrylamide + pH gradient | Isoelectric point (pI) | Proteins | High-resolution protein separation |
| Capillary electrophoresis (CE) | Capillary (no gel or coated) | Charge-to-size ratio ± sieving | DNA, proteins, small ions | DNA sequencing, clinical diagnostics |
4. Direction of Migration
- Positively charged molecules → migrate to cathode (−)
- Negatively charged molecules → migrate to anode (+)
- Most nucleic acids (DNA/RNA) → negatively charged (phosphate backbone) → move toward anode
- Proteins → direction depends on buffer pH relative to their isoelectric point (pI)
5. Common Visualization Methods
| Analyte | Common stain/detection | Appearance |
|---|---|---|
| DNA / RNA | Ethidium bromide, SYBR Safe/Green, GelRed | Fluorescent orange-red under UV |
| Proteins (SDS-PAGE) | Coomassie Brilliant Blue R-250, Silver stain | Blue bands, very sensitive silver |
| Glycoproteins | Periodic acid–Schiff (PAS) | Magenta |
| Enzyme activity | Activity staining (zymography) | Colored/cleared zones |
6. Immune Electrophoresis
Immune electrophoresis is a powerful hybrid technique that combines electrophoretic separation with immunodiffusion to identify specific proteins within complex mixtures like human serum.
Principle: Proteins are first separated by their electrophoretic mobility in an agarose gel. A trough is then cut parallel to the separation path and filled with specific antibodies. As both diffuse through the gel, they meet and form visible precipitin arcs at the point of optimal concentration.
Comparison Table
| Feature | Standard Electrophoresis | Immune Electrophoresis |
|---|---|---|
| Separation Basis | Charge, size, shape | Charge, size + Immunological specificity |
| Detection | Dye-based staining | Precipitation arcs (Antigen-Antibody) |
| Specificity | Lower | High (identifies specific proteins) |
Clinical & Forensic Applications
- Protein Profiling: Identifying abnormal protein patterns in serum (e.g., Multiple Myeloma).
- Immunodeficiency Screening: Detecting deficiencies in specific immunoglobulin classes (IgG, IgA, IgM).
- Forensic Characterization: Identifying specific biological proteins in complex trace evidence.
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