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The isoelectric point (pI) is the pH at which a particular molecule carries no net electrical charge. Biological amphoteric molecules such as proteins contain both acidic and basic functional groups. Amino acids that make up proteins may be positive, negative or neutral according to the environment pH, and thus give a protein its overall charge. 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, thus, be detected by isoelectric focusing (IEF).In IEF, a slab of polyacrylamide or agarose gel that contains a mixture of ampholytes is used to create a pH gradient when subjected to an electric field. During IEF analysis, molecules to be focused are distributed over the medium with established pH gradient. An electric current passes through the medium and creates the "positive" anode end and "negative" cathode end. Negatively charged molecules migrate through the pH gradient in the medium toward the "positive" end while positively charged molecules move toward the "negative" end.
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