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 |