ISOELECTRIC FOCUSING

Introduction

Isoelectric focusing can be defined as an analytical tool powerful enough to separate proteins. The isoelectric point is different for different protein molecules and is technically used for separating various protein molecules. The high performance is being ensured through previously set isoelectric point standards or pI. Compounds having lower molecular weight have been successfully developed and examined in IEF capillary or IEF electrophoresis. It is a variant of zone electrophoresis that is being performed on proteins using gel. The fact that surrounding pH contributes to overall charge of a significant molecule is of great advantage.

Discussion

Isoelectric Focusing Principle

The main principle of isoelectric focusing is that two different proteins having different isoelectric points migrate in pH gradient and presence of electric field till total charge of a protein gets to zero. The migration stops as the condition is achieved. Protein gets immobilized in the respective pH gradient while approaching their pI. The gel is then stained and results are documented. After the separation of protein 2D gel electrophoresis is used for further separation of protein with respect to their mass. When net charge of protein gets to zero but cannot be further moved in presence of an electric field. Protein remains in its position due to gradient force and this phenomenon is known as focusing. Reduction in sample or increase in voltage will result in appearance of more clear bands. Application of voltage results in the generation of heat but implication of thin gels and thermostatic circulator will result in sharp focussing. The minimum pI is used for estimating separation. The charge of protein is the sum of its negative and positive charges. These charges are dependent on the pH of their surroundings. The pI of a protein is low if the acidic group is more than the basic group. The pI of a protein will be high if the basic group is more than the acidic group. Proteins get a positive charge when the surrounding pH is below their pI. Protein gets a negative charge when pH surrounding the protein is higher than the protein’s pI.

Isoelectric Focusing Electrophoresis

Isoelectric focusing is generally used to separate proteins of the same molecular mass. The main principle is that physical characters of a protein are being exploited for their separation.  Isoelectric focusing electrophoresis can also be termed as two dimensional or 2-D electrophoresis. The first process is dependent on their charge and the second process is determined through mass.  While processing with the first step the cell is first denatured. Using a high concentration of urea, that is almost 8M. It is then layered on a glass tube with a polyacrylamide solution of ampholytes. Ampoholyes is a mixture of solution continuing polycationic and polyanionic molecules. These ampholytes will separate during the application of the electric field and a continuous gradient is formed. The high polycationic molecules will collect at one end and the most polyanionic molecules will collect at the other end. The establishment of a pH gradient is stimulated by these ampholytes. Proteins will now migrate through these gradients until the isoelectric point is reached. At this point, the protein acquires zero charges. This is known as isoelectric focusing. The Acrylamide gel is used for carrying out isoelectric focussing. Acrylamide is present in gel along with non-ionic detergent and urea. It helps in denaturing and maintaining the protein’s solubility. 

Isoelectric Focusing 

In isoelectric focusing, it is already known that net charge of protein gets zero and it gets focused in the gel due to the surrounding pH gradient. The net charge is zero indicates that positive and negative charges are balanced and repulsive electrostatic forces get reduced. The precipitation and aggregation of protein are caused due to attraction forces that predominate. When the pH of a solution is above the protein’s pI, the protein gets a negative charge and experiences a repulsive force from the surroundings. When the pH is lower the protein pI, the protein will be positively charged and will also experience repulsive force from surrounding molecules. However, at an isoelectric point, the negative and positive charges cancel out. The pH ranges from 4-6. Mineral acids such as sulfuric acid and hydrochloric acid are used for precipitation. One Of greatest advantages of such precipitation is that proteins can get irreversibly denatured. Therefore containment proteins are generally used for isoelectric point precipitation. Target proteins are not specifically used for such purposes. An example of isoelectric precipitation is casein precipitation during cheesemaking or during the process of producing sodium caseinate. The mechanism that underlies the concept of precipitation is the alteration of solvation potential. When the reagent has been added the solubility of the solute gets reduced. In the industry of biotechnology, protein precipitation technique is generally used for containments elimination in blood components.  

Conclusion

The Protein’s surface contains several hydrophilic and hydrophobic amino acid residues. These residues are responsible for protein solubility in aqueous solutions. The presence of hydrophobic protein is generally found in the globular core of protein. But some of these residues can be found on the surface of the protein as patches. Proteins containing higher hydrophobic residues on the surface will be less soluble in an aqueous solvent. The protein solubility gets increased when there is an interaction between solvent ionic groups and charged polar surface residues. The isoelectric point is different for different protein molecules and is technically used for separating various protein molecules.  Isoelectric focusing is a useful technique for separation of the protein and is considered the analytical tool. Precipitation of the protein can also be done using isoelectric points and focusing.