Isoelectric focusing

[1][2] It is a type of zone electrophoresis usually performed on proteins in a gel that takes advantage of the fact that overall charge on the molecule of interest is a function of the pH of its surroundings.

The technique is capable of extremely high resolution with proteins differing by a single charge being fractionated into separate bands.

At this point the molecule no longer has a net electric charge (due to the protonation or deprotonation of the associated functional groups) and as such will not proceed any further within the gel.

The gradient is established before adding the particles of interest by first subjecting a solution of small molecules such as polyampholytes with varying pI values to electrophoresis.

The method is applied particularly often in the study of proteins, which separate based on their relative content of acidic and basic residues, whose value is represented by the pI.

When a sample (a mixture of peptides or proteins) is injected in the capillary, the presence of the electrical field and the pH gradient separates these molecules according to their isoelectric points.

The multi-junction IEF system has been used to separate tryptic peptide mixtures for two-dimensional proteomics[9] and blood plasma proteins from Alzheimer's disease patients for biomarker discovery.