Isoelectric focusing

As isoelectric focusing or shortly IEF is an electrophoretic separation of proteins in a gel (IEF-PAGE), in a capillary , or in a liquid film due to its relative content of acidic and basic amino acid residues , respectively.

principle

Depending on the pH value of the surrounding medium, the amino acids of a protein carry different, positive or negative charges (see article ampholytes ). The sum of all charges on a protein is zero at a certain pH - its isoelectric point ( pI ). If a protein molecule reaches a point in the medium with this pH value in an electric field, its electrophoretic mobility disappears there , so that it remains there. Since all the molecules of one type of protein collect at the same point in the medium, this is known as focusing .

execution

In IEF-PAGE, a protein mixture is introduced into a carrier gel (usually polyacrylamide ) in which a pH gradient has been established beforehand. This gradient is established either electrophoretically by means of carrier ampholytes that are freely movable in the electrical field or via immobilized (i.e. covalently connected to the carrier gel ) charge carriers. An electrical voltage is then applied to the gel. Each protein now moves in the electric field due to its own charge until the pH value of the carrier gel corresponds to its pI. A protein that diffuses away from this gel region receives a net charge again due to the change in the pH value and moves again into the area of the gel whose pH value corresponds to its pI. The electric field thus concentrates ('focuses') every single protein in the gel in the pH range of its specific pI.

commitment

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

Web links

FischDB (fish database) - contains numerous IEF analyzes

swell

↑ Optimized continuous flow electrophoresis. Weber G, Bocek P .; Electrophoresis; 1996 Dec; 17 (12): 1906-10, PMID 9034773 .
↑ The current state of two-dimensional electrophoresis with immobilized pH gradients: Görg A, Obermaier C, Boguth G, Harder A, Scheibe B, Wildgruber R, Weiss W. Electrophoresis. 2000 Apr; 21 (6): 1037-53, PMID 10786879 .
↑ High-resolution two-dimensional electrophoresis with isoelectric focusing in immobilized pH gradients. Westermeier R, Postel W, Weser J, Görg A .; J Biochem Biophys Methods. 1983 Dec; 8 (4): 321-30, PMID 6663005 .

[1] Optimized continuous flow electrophoresis. Weber G, Bocek P .; Electrophoresis; 1996 Dec; 17 (12): 1906-10, PMID 9034773 .

[2] The current state of two-dimensional electrophoresis with immobilized pH gradients: Görg A, Obermaier C, Boguth G, Harder A, Scheibe B, Wildgruber R, Weiss W. Electrophoresis. 2000 Apr; 21 (6): 1037-53, PMID 10786879 .

[3] High-resolution two-dimensional electrophoresis with isoelectric focusing in immobilized pH gradients. Westermeier R, Postel W, Weser J, Görg A .; J Biochem Biophys Methods. 1983 Dec; 8 (4): 321-30, PMID 6663005 .