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Study Notes: The Electrophoretic System

Electrophoretic apparatus comes in a number of varieties and sizes but each system is essentially an electrical system comprising upper and lower buffer tanks (often) connected to a gel sandwiched between two plates of glass. The apparatus is watertight. A current is applied to the gel via the upper tank (negative) and the lower tank (positive).

The electric field causes the samples (introduced into wells in the top of the gel) to migrate towards the bottom chamber as long as all the species of interest carry a (negative) charge. In SDS-PAGE this is accomplished by the SDS, which attaches to the protein imparting an overall (negative) charge. The upper tank, the gel and the bottom tanks are resistors to the flow of current but are designed so that the gel generates the majority of the resistance in the circuit. The voltage drop across the gel due to this resistance provides the motive force to drive the buffer and sample ions through the gel.

An image of a gel electrophoresis apparatus, containing 2 diagrams.  The first shows an old style apparatus, with the upper and lower tanks connected by a panel that contains the gel.  The diagram on the right shows the electrical circuit and flow of molecules in the circuit.

Ohm’s Law in electrophoresis
The voltage V, the current I and the resistance R, are related by Ohm’s Law:

V = IR

that is, an increase in voltage will produce an increase in current through a given resistor (in this case the gel).

A variation to Ohm’s law describes how small changes in current, I, produce large changes in the expenditure of power P in the circuit:

P = IV = I2R

This is important as small changes in current lead to changes in temperature and temperature can be a problem in electrophoresis. The vast majority of current in electrophoresis is due to migration of buffer ions and sample molecules (remember that they are charged). Thus as voltage is applied:

Cations (+) migrate to the (negative) electrode in the upper chamber
Anions (-) migrate to the (positive) electrode in the lower chamber.

Several factors must be correct for electrophoresis to proceed:

  • Sample molecules must be negatively charged
  • Upper and lower tanks and the gel itself must contain adequate amounts of (+) and (-) ions
  • System must be leak proof with regard to liquid and electric current
  • Temperature must be controlled (remember that resistance means heat generation in an electrical system).

Remember that the current is via the flow of ions in the gel. Very mobile (small) buffer ions create a more conductive buffer because they migrate faster and this excessive current flow will lead to excessive heat generation. If uncontrolled, this may lead to convection currents in the gel, solution evaporation and melting of agarose gels.

Heat generation is controlled by:

  • use of bulky organic ions such as Tris base or glycine (check glossary for definitions of these terms) in the buffers
  • use of a power pack that maintains a constant current (a rise in temperature would normally lead to a rise in current and a further rise in temperature but constant current conditions stop this from happening) although in modern equipment constant voltage can also be used
  • use of a large stirred lower buffer tank that acts as a heat sink.

 

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