Principle of Isoelectric Focusing (IEF)

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Isoelectric Focusing or IEF is a method of separating proteins according to their Isoelectric points in a pH gradient. Isoelectric point denoted as pI is defined as the pH at which protein carry no net charge, or pH at which protein become immobile in an electric field.

One of the best method for characterizing proteins, because protein separation /purification can be easily done if Isoelectric point of a protein is known.

Isoelectric Focussing Principle

The resolution of Isoelectric Focussing is very high. In normal electrophoretic methods pH between anode and cathode remains constant but in Isoelectric focussing , a pH gradient is arranged.when pH of protein is below its pI proteins become positively charged and it will migrate towards cathode. because of the pH gradient charge of the protein molecule changes while moving forward, there will be a point at which net charge of the protein becomes zero this point is called Isoelectric point. when a protein mixture or a single protein is run under an electric field at specific pH the protein stops moving.