Seed Dispersal: Definition, Types, Importance, Adaptations & Examples

 Isoelectric focusing/ Electro focusing

Electrophoresis along the gradient of pH values in the supporting medium is called electro focusing. In this method, electric current forces the protein molecules to reach the isoelectric point in the pH gradient. There is no further movement of proteins after reaching the same pH as their isoelectric point. Hence, this method is also called isoelectric focusing (IEF). The IEF is very useful for the separation of closely related proteins having the same size and weight but different isoelectric points 

Principle

Proteins contain many positively and negatively charged groups on their surface. Therefore, each protein has some net ionic charge. Solutions also have some ionic charge depending on the cations and anions in them. This ionic charge of solutions varies with pH values. The pH at which the ionic charge of a solution is equal to the ionic charge of a protein is called isoelectric point of the protein. At the isoelectric point, the number of negative charges on the protein is equal to the number of positive ions in the solution. So, the net electric charge is zero. Since the electrical potential difference is zero, proteins do not move further while moving across the pH gradient. Here, proteins are focused to their isoelectric pH of the medium. Proteins exist as cations at pH values below the isoelectric point. They exist as anions at pH range above the isoelectric point. If proteins are allowed to move along a pH gradient across the gel under the influence of electric current, they move to the isoelectric pH and they do not move further due to electric current. Many closely related proteins differ in their isoelectric pH because of the presence of different acidic and basic groups. Hence, such proteins can be separated by electro focusing. The apparatus consists of a lower buffer tank, an upper buffer tank and a glass tube. The glass tube is fixed in between the two tanks. One electrode is fixed in each buffer tank

Procedure

1. A short glass tube with open ends is taken and cleaned well. 
2. The lower end of the tube is closed with a cork.
3. A sucrose density gradient column with pH gradient is prepared in the tube.
4. Ampholyte containing 1.94g acrylamide, 0.06g bisacrylamide, 5g sucrose, 0.25ml riboflavin solution and 40ml of water is prepared. The ampholyte solution is poured slowly along the wall of the tube until it reaches the level of the column inside.
5. The tube is kept undisturbed for 3hrs for the polymerization of polyacrylamide gel with pH gradient. 
6. After polymerization of the gel, the cork at the base is removed.
7. A sample containing proteins is poured on the top of the pH gradient column.
8. The tube is attached with two buffer reservoirs.
9. A solution with more acidic pH is filled in one buffer reservoir and another solution with more alkaline pH is filled in the other buffer reservoir.
10. Electrodes are connected and the electrophoresis unit is switched on.
11. Proteins move along the gel under the influence of electric current. After reaching the isoelectric pH, there is no further movement of the protein. This movement can be seen by the movement of bromophenol blue dye added to the protein solution.
12. After disconnection, the particular band is taken from the gel to isolate the protein.

Applications

1. The IEF is very useful for the isolation and purification of proteins having the same mass but different isoelectric points.
2. It is a sensitive technique to resolve closely related proteins.
3. Widely used for separation and identification of serum proteins.
4. Used in food and agricultural industries, forensic and human genetics laboratories.
5. Used in enzymology, immunology and membrane biochemistry.
6. Used in 2D Gel electrophoresis is an application of IEF.