What is the significance of isoelectric focusing in protein separation?

Isoelectric focusing is a technique that separates proteins based on their isoelectric point (pI), which is the pH at which a protein carries no net electrical charge. In this method, a pH gradient is established in a gel or solution, and proteins are applied to this medium. When an electric field is applied, proteins migrate until they reach the point in the gradient where the pH matches their pI. At this point, because they have no net charge, they stop moving. This makes isoelectric focusing particularly effective for resolving proteins with very similar sizes or molecular weights but different pI values.

The significance of isoelectric focusing lies in its ability to provide high-resolution separation of proteins, allowing for the study of complex mixtures, such as those found in biological samples. This technique also aids in the identification and characterization of proteins, as the pI can provide insights into the protein's structure and function.