CIEF (Capillary Isoelectric Focusing) Analysis

CIEF (Capillary Isoelectric Focusing) is a high-resolution separation technique based on the difference in isoelectric points (pI) of amphipathic molecules such as proteins. Its core principle is to use an electric field to drive an amphoteric electrolyte to form a continuous pH gradient within a capillary, causing sample molecules to migrate to the pH region corresponding to their pI (with zero net charge) and focus into a narrow band. In the experiment, the sample is mixed with the amphoteric electrolyte and injected into the capillary. After applying a high voltage, the proteins migrate directionally according to the relationship between their local pH and their own pI: when the ambient pH is lower than the pI, they migrate towards the negative electrode with a positive charge, and vice versa, until they reach the pI position and stop. After focusing, pressure or electromigration pushes the band through a detection window (such as ultraviolet absorption), generating an electrophoretic pattern for analyzing the pI of each component. This technology requires no labeling, can distinguish proteins with a pI difference of only 0.01, and requires only nanoliter samples. It combines high sensitivity with automation and is widely used in fields such as monoclonal antibody charge heterogeneity analysis, biomarker screening, vaccine quality control, and post-translational modification research. Deamidation, aspartic acid and glycine alterations to form succinimide, C-terminal lysine heterogeneity, glutamate and glutamine conversion, disulfide bond oxidation, and the degree of sialylation glycosylation can all cause changes in the isoelectric point of proteins.