Proteins are amphoteric molecules, which contain both acidic and basic amino acids. Amino acids that make up proteins may be positive, negative or neutral, depending on the environment pH, and thus give a protein its overall charge. The isoelectric point (pI) is the pH at which a particular molecule carries no net electrical charge. Because the specific mutations, truncation or post-translational modifications can change the pI of the natural proteins. The net charge on the molecule is affected both by its own pI, and pH of the surrounding environment, And the proteins can become more positively or negatively charged due to the gain or loss, respectively, of protons (H+). At a pH below their pI, proteins carry a net positive charge; above their pI they carry a net negative charge; equal to their pI, proteins have no net charge and thus do not migrate further in the electric field.
The isoelectric point of proteins can be detected by isoelectric focusing (IEF). When proteins move towards the pole opposite of its charge, they get immobilized in the pH gradient as they approach their specific pI. As a result, the proteins become focused into sharp stationary bands with each protein positioned at a point in the pH gradient corresponding to its pI. In IEF, proteins only differing by a single charge could be fractionated into separate bands, giving this technique extremely high resolution.
Capillary isoelectric focusing (cIEF) Technology
Capillary isoelectric focusing allows the separation of protein/peptide mixtures, protein glycoforms and other charged variants, based on their isoelectric point (pI). A voltage is applied to the anolyte and catholyte tanks, establishing a pH gradient through the capillary. Proteins that are applied in the field can be separated based on their net charge, and become immobilized when they reach a pH, which equals to their pI.
Compared to conventional isoelectric focusing (IEF) in gels, cIEF allows for higher resolution, faster sample analysis with 10 fold lower limit of detection. What’s more, cIEF allows real-time observation and eliminate sample degradation during the lengthy mobilization phase in conventional isoelectric focusing.
In Creative Proteomics, capillary isoelectric focusing, cIEF, an updated version of Capillary electrophoresis-based technique is utilize for pI determination, which displays superior resolution and accuracy, reproducibility and high throughput; and Ion exchange chromatography, which can separate the protein molecules depending on their net charge, is used to monitor charge variants of protein therapeutics, to evaluate comparability between different lots.
Capillary isoelectric focusing (cIEF) is a high-resolution analytical technique. Our capillary electrophoresis service allows you to determine the isoelectric point of a protein quickly and accurately.
Application of determination of PI:
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