soelectric focusing (IEF) is a technique used to separate molecules based on their isoelectric point (pI), which is the pH at which a molecule has no net charge. In IEF, a mixture of charged molecules, such as proteins or peptides, is applied to a gel matrix containing a pH gradient. An electric field is then applied across the gel, causing the charged molecules to migrate to their isoelectric point, where they become electrically neutral and stop moving. The pH gradient in the gel can be created using different methods, such as immobilized pH gradient (IPG) strips or carrier ampholytes. IPG strips are precast gels that contain a pH gradient formed by covalently linking a series of buffering molecules of different pK values to the gel matrix. Carrier ampholytes, on the other hand, are small molecules that migrate in the electric field and create a pH gradient by buffering the surrounding solution. During IEF, the charged molecules move through the pH gradient in the gel, reaching their isoelectric point and becoming immobilized at that point. The separation of the molecules is based on their differences in pI values, with molecules with higher pI values moving towards the anode and those with lower pI values moving towards the cathode. The separation is visualized by staining the gel with a suitable dye or by transferring the separated molecules onto a membrane for further analysis. IEF has many applications in protein analysis and purification, including the separation of protein isoforms, identification of post-translational modifications, and characterization of protein-protein interactions. It is a powerful tool for identifying and characterizing proteins in complex mixtures and is commonly used in proteomics research.