Electrophoresis & General technique for electrophoresis.

Electrophoresis & General technique for electrophoresis.

Electrophoresis

What is Electrophoresis ?

• The movement of charged particles such as molecules or ions through an electrolyte under the influence of electric current is called electrophoresis. The electric field is applied to a solution by placing two oppositely charged electrodes in the solution.

• Depending on the nature of charges, the molecules move through the solution towards the electrode of the opposite charge.

• The positively charged particles, the cations, move towards the negatively charged electrode (cathode); and negatively charged particles, the anions, move towards the positively charged electrode (anode).

• At a fixed pH, the rate of migration of particles with similar charge will depend on the magnitude of charges carried on them and molecular weight: or charge/ size ratio.

Because of these variations in migration, it is possible to separate a complex mixture such as plasma proteins into a number of fractions or zones. This is called zone electrophoresis.

Isoelectric Focusing

• A large molecule like protein has different groups such as carboxyl- (negatively charged) and amino(positively charged) groups. The sum total of the charges on such different side groups decides the net charge on the molecule.

• Since the electric charge varies with pH, the pH at which a particular protein has net charge equal to zero is called its isoelectric point.

• In the technique of isoelectric focusing, an electrolyte is used which produces a gradual increase in the pH from anode to cathode on the passage of electric current.

•  If a complex mixture of substances is electrophoressed in such a solution, the fractions become arranged in order of the increasing pH of their isoelectric points. 

• In a solution of gradually increasing pH, the molecules migrate towards the anode or cathode until they arrive at the point at which the pH is that of their isoelectric point.

Electro-endosmosis

• Electro-endosmosis is a phenomenon which affects the movement of the molecules of the buffer fluid itself under the influence of the electric current. At an appropriate pH, the buffer molecules are attracted towards the cathode (-).

• The negatively charged protein molecules move towards the anode (+), but the electro-osmotic flow of the buffer fluid pushes the proteins back in the opposite direction towards the cathode.

• Under such conditions, the slowest moving proteins such as gamma-globulins which have a very small negative charge, are carried towards the cathode side of the point of application.

This phenomenon is used for the detection of hepatitis B antigen and antibody in patient's serum (see Microbiology section).

Equipment and Materials

• The equipment and materials needed for electrophoresis generally consist of a buffersystem, a sample applicator, a solid medium such as paper. cellulose acetate, polyacrylamide gel, stareh gel or agar gel, an electrophoresis tank and a power supply. 

Stains and washing solutions may be necessary for staining and elution of separated fractions. 

General technique for electrophoresis 

1. Place a hydrated support medium such as cellulose acetate or agar gel into the electrophoresis chamber.

2. Place the buffer of appropriate pH in the electrode chambers of the electrophoresis tank.

3. Keep the support medium in contact with the buffer, for example, by using paper wicks.

4. Apply the sample to the support medium. 

5. Conduct electrophoresis for a determined length of time using either constant voltage or constant current. 

6. Remove the support from the chamber immediately and dry or place it in a fixative to avoid diffusion of sample components. 

7. Stain the components, wash the excess dye and dry or place in a clearing agent.