The method is applied in the study of proteins, which separate based on their relative content of acidic and basic residues, whose value is represented by the pI. The gradient is initially established before adding the particles of interest by first subjecting a solution of small molecules such as polyampholytes with varying pI values to electrophoresis. At this point the molecule no longer has a net electric charge (due to the protonation or deprotonation of the associated functional groups) and as such will not proceed any further within the gel. As a particle moves towards the pole opposite of its charge it moves through the changing pH gradient until it reaches a point in which the pH of that molecules isoelectric point is reached. The negatively charged molecules migrate through the pH gradient in the medium toward the 'positive' end while positively charged molecules move toward the 'negative' end. An electric current is passed through the medium, creating a 'positive' anode and 'negative' cathode end. Molecules to be focused are distributed over a medium that has a pH gradient (usually created by aliphatic ampholytes). This technique is capable of extremely high resolution with proteins differing by a single charge being fractionated into separate bands. As a result, the proteins become focused into the sharp stationary bands with each protein positioned at a point in the pH gradient corresponding to its pI. At this point it has no net charge and so migration ceases. As it migrates, however, the charge will decrease until the protein reaches the pH region that corresponds to its pI. A protein that is in a pH region below its isoelectric point (pI) will be positively charged and so will migrate towards the cathode. Isoelectric focusing is a type of zone electrophoresis, and it is usually performed in a gel, that takes advantage of the fact that a molecule's charge changes with the pH of its surroundings.

For a protein with many basic amino acids, the pI will be high, while for an acidic protein the pI will be lower. Isoelectric point (pI): The pH at which the net charge on the protein is zero.

This process can successfully separate proteins that have a difference in net charge greater than or equal to 1. a protein band that forms at a given pH can then be removed and analyzed further. In other words, each protein will move until it reaches a position in the gel at which the pH is equal to the pI of the protein. The proteins will move along the gel until they reach their isoelectric points. Then the sample is put onto the gel and a voltage is applied. The pH gradient is made by adding polyampholytes, which are multi-charged polymers, with different pI into the gel. A gel with a pH gradient is used as the medium. Isoelectric focusing is a separation technique which separates peptides according to their isoelectric point, or how acidic and basic their residues are. Isoelectric point, also called the pI of the protein, is the pH at which the net charge of the protein is zero. Each charged particle will migrate in a pattern determined by its particular property due to changes in speed and direction, allowing for the separation of biomolecule components with similar properties.Isoelectric Focusing Negatively charged particles such as nucleic acids gravitate toward the anode, while the positively charged particles toward the cathode. Depending on the mass and net charge of each particle in the solution, ionized biomolecules will migrate at different rates when exposed to an electric field. When charged molecules are placed in an electric field, they travel in the opposite direction of the positive or negative pole. A solution containing biomolecules will have either positively or negatively charged ions depending on the pH. Principle of Gel Electrophoresis The principle behind electrophoresis is the observation that the majority of biomolecules exist as electrically charged particles with ionizable functional groups. Gel electrophoresis is one of the laboratory methods for separating DNA, RNA, or protein molecules based on their electric charge or size. In 1948, Arne Tisselius received Novel Prize in Chemistry for his contributions to the electrophoretic technique. In the 1930s, Swedish biophysicist Arne Tisselius developed electrophoresis while researching blood proteins. Electrophoresis is a chemical process in which an electric charge in a solution flow toward an opposing electrode.

Isoelectric point measure