Isoelectrofocusing

Isoelectrofocusing (IEF) is a family of techniques that separate amphoteric molecules, principally proteins, based on their isoelectric point (pI)—the pH at which the molecule carries no net electric charge. In a pH gradient, proteins migrate in an electric field until they reach the pH equal to their pI, at which point net charge is zero and migration stops.

The pH gradient can be formed with carrier ampholytes in gel or solution or, more stably, via immobilized pH gradients on gels (IPG strips). In carrier ampholyte-based IEF, a mixture with buffer salts and ampholytes creates a continuous gradient; proteins are loaded and the gel is subjected to an electric field. In IPG-IEF, the gradient is fixed by immobilized buffering groups, producing greater reproducibility.

Mediums for IEF include gel-based systems that use polyacrylamide or agarose matrices, and capillary IEF (CIEF) that employs capillaries for high resolution and small sample volumes. In practice, IEF is often the first dimension of two-dimensional gel electrophoresis (2-DE), followed by SDS-PAGE to separate by molecular weight.

Sample preparation commonly includes denaturants (urea or thiourea), detergents (CHAPS), reducing agents (DTT), and buffering components. Detection is via staining or fluorescence; proteins resolved along the pI axis appear as discrete bands or spots.

Applications span proteomics, analysis of protein isoforms and post-translational modifications, clinical chemistry, and purification processes. Limitations include gradient instability, sample interference, and limited ability to resolve very acidic or basic proteins.