Principle of Electrophoresis

• Movement of dispersed, charged particles relative to a fluid under the influence of spatially uniform electric field. Used in the separation of DNA, RNA, or proteins based on the molecular size and net electrical charge.
• Some forces happen when you apply it in an electric field.
• It is the attraction or repulsion between charged particles.

  Electrostatic Force (Coulomb Force)
• Resistance to motion when the surface of one object comes in contact woth another.

  Friction Force
• Force exerted by the medium on particles in a direction opposite to particle movement (buffer and gel).

  Electrophoretic Retardation Force
• If the forces are not equal, molecule will still move, but it is moving because of an electrostatic force. The forces should be equal.
• Formula for Electrostatic Force (Fe).

  Fe=E x q

  E=electric force (V/m)

  q=charge of particles (C)
• Formula for Drag Force (Fd)?


  Fd = f x v

  F = frictional coefficient

  V=velocity of the particle (m/s)
• Fe = ?

  Fd
• Starting electrophoresis equates these forces to have net movement of the particles based SOLELY on size and charge.
• Solute response to the electric field (u)?

  Electrophoretic Mobility
• Calculates how fast this particle will go and which direction will it go?

  Velocity
• Formula for Electrophoretic Mobility?

  u = q/f = v/e
• Formula for Velocity of the Particle?

  v = q x E / f
• Factors Affecting Electrophoresis?

  Net Charge

  Molecular Size

  Molecular Shape

  Applied Voltage

  Buffer

  Gel
• Factor Affecting Electrophoresis: Charges will migrate to the OPPOSITE pole (cation to anion).

  Net Charge
• Factor Affecting Electrophoresis: Smaller molecules will travel farther; Larger molecules will be retained?

  Molecule size
• Factor Affecting Electrophoresis: Denatured DNA will ligrate more predictability according to size.

  Molecule Shape
• Factor Affecting Electrophoresis: Voltage = current resistance (V=IR), increase in voltage = increase in current.

  If you increase in voltage, current and run will be faster. Because it is fast, heat will also accumulate faster. Gel is a gel, that can melt. Too high voltage = too high current = gel will melt, damaging the samples.

  Applied Voltage
• Factor Affecting Electrophoresis: Composition and properties of the buffer affects electrophoretic mobility?

  Buffer
• Factor Affecting Electrophoresis: Pore size of the gel will determine the ease of movement of different sized macromolecules.

  Gel