Isoelectric focusing

Isoelectric focusing IEF as a short or an electrophoretic separation of proteins in a gel (IEF -PAGE), in a capillary, or in a liquid film is known, due to their relative levels of acidic and basic amino acid residues.

Principle

Depending on the pH of the surrounding medium carrying the amino acids of a protein different positive or negative charges ( see Article ampholytes ). The sum of the charges of a protein at a particular pH - value - its isoelectric point (pI) - zero. Where a protein molecule in an electric field at a location of the medium at that pH, there disappears its electrophoretic mobility, so that it remains lying there. Since collect all the molecules of a protein places at the same point of the medium, it is called focusing.

Implementation

In IEF -PAGE, a protein mixture is introduced in a carrier gel (usually polyacrylamide), in the above, a pH gradient was established. The establishment of this gradient is carried out either by means of electrophoresis in the electric field of freely moving carrier ampholytes or immobilized ( ie, covalently attached to the carrier gel ) charge carriers. Then an electrical voltage is applied to the gel. Each protein is now moving in the electric field due to its inherent charge so far, until the pH of the carrier gel corresponds to its pI. A protein diffuses away from the Gelregion obtained by changing the pH back a net charge, and moves back into the area of the gel, the pH corresponds to its pI. Thus, the electric field concentrates ( ' focused ') of each individual protein in the gel in the pH range of its specific pI.

Use

The technique of isoelectric focusing is used to ( analytical ) Determination of pI of proteins and for ( preparative ) charge-dependent fractionation of protein mixtures. With the introduction of immobilized pH gradient (IPG ) by immobilines, the reproducibility of individual focusing runs has improved significantly. Immobilines are weak acids or bases that have a defined pK value and bond covalently with the gel matrix. This development was the prerequisite for the optimization of high-performance protein analysis techniques such as 2D gel electrophoresis. IEF is used here to electrophoretic protein separation in the first dimension.