Electrophoresis

ELECTROPHORESIS

In clinical laboratories, various devices are used based on the electrophoretic principle. These devices are used for the following applications.

Ø To measure the quantity of protein in plasma, urine, etc.

Ø To separate enzymes into their components is enzymes. 

Ø To identify antibodies.

Basic principle

Electrophoresis is defined as the movement of a solid phase with respect to a liquid. The buffer solution is used to carry the current and to maintain the pH value of the solution as a constant one during the migration.

In this title, zone electrophoresis is explained. In this technique, the sample is applied to the medium and under the effect of the electric field, group of particles that are similar in charge, size, and shape migrate at the same rate. So the particles are separated into zones.

Factors Affect the Speed of Migration

Magnitude of charge:

The mobility of a given particle is directly related to the net magnitude of the particles charge. Mobility is defined as, the distance in cm, a particle moves in unit time per unit field strength.

Ionic Strength of Buffer

If the buffer is more concentrated then the migration of the particles is slow. Because, if greater the proportion of buffer ions present, then greater the proportion of the current they carry.

Temperature:

Mobility is directly related to temperature. Heat is produced when the current flows through the resistance of the medium. So, the temperature of the medium is increased and resistance is decreased. Finally, the rate of migration is increased.

The water is evaporated from the surface of the medium due to heat. So, the concentration of particle is increased. Finally the rate of migration is increased. When the gel is used as a medium; this heat will create a problem. So, for this medium, constant current sources are used to minimize the heat production.

Time: The distance of migration is related to the time period during which electrophoresis takes place.

Types of Support Media:

Cellulose acetate, starch gel and sucrose are used as support media in various electrophoretic applications. We can see the cellulose acetate electrophoresis in the following sections.

Cellulose Acetate Electrophoresis

Cellulose acetate strip is saturated with the buffer solution and placed in the membrane holder. It is otherwise known as bridge. The two ends of the bridge are placed in the cuvette in which buffer solution is available.

The sample for each test is placed on the strip at a marked location. Then, the constant electric potential(250 V) is applied across the strip 4 – 6 mA of initial current is obtained .After 15-20mins, the electric voltage is removed, then, migrated protein band is stained with buffer and it is dried in preparation for densitometry.

The membrane is placed in the holder of densitometer. The path of the migration of one of the specimen is scanned. The low voltage output is amplified and recorded using x-y recorder.