The first dimension in a 2-D gel electrophoresis experiment involves the separation of proteins according to their isoelectric point (pI) by isoelectric focusing (IEF). It offers the highest resolution of all electrophoresis techniques (Westermeier 2004)1.
IEF works by applying an electric field to protein within a pH gradient. The proteins separate as they migrate through the pH gradient in response to the applied voltage. When a protein reaches a pH value that matches its pI, its net electrical charge becomes neutral, and stops migrating. In this way, each protein in a sample becomes "focused" according to its pI. IEF can be performed using two techniques: immobilized pH gradients (IPG) with ampholytes covalently bound to a gel, or carrier ampholytes that migrate through a gel to generate the pH gradient. This section provides technical details to perform successful IEF using IPG strips
When a protein is placed in a medium with a pH gradient and subjected to an electric field, it will initially move toward the electrode with the opposite charge. During migration through the pH gradient, the protein will either pick up or lose protons. As it migrates, its net charge and mobility will decrease and the protein will slow down. Eventually, the protein will arrive at the point where the pH gradient is equal to its pI. There, being uncharged, it will stop migrating (see figure below). If this protein should happen to diffuse to a region of lower pH, it will become protonated and be forced back toward the cathode by the electric field. If, on the other hand, it diffuses into a region of pH greater than its pI, the protein will become negatively charged and will be driven toward the anode. In this way, proteins condense, or are focused, into sharp bands in the pH gradient at their individual characteristic pI values.
This system designed to simultaneously run up to 12 immobilized pH gradient (IPG) strips in 12 independently programmed lanes, which allows users to confidently run 12 different conditions at one time. Although many available IEF systems have multiple lanes, all but the PROTEAN i12 system depend on a single power supply, allowing only one set of conditions to be run at a time.
IEF can be run under either native or denaturing conditions. Native IEF retains protein structure and enzymatic activity. However, denaturing IEF is performed in the presence of high concentrations of urea, which dissociates proteins into individual subunits and abolishes secondary and tertiary structures. Whereas native IEF may be a more convenient option because it can be performed with a variety of precast gels, denaturing IEF often offers higher resolution and is more suitable for the analysis of complex protein mixtures.
Focusing is a steady-state mechanism with regard to pH. Proteins approach their respective pI values at differing rates, but remain relatively fixed at those pH values for extended periods. By contrast, proteins in conventional electrophoresis continue to move through the medium until the electric field is removed. Moreover, in IEF, proteins migrate to their steady-state positions from anywhere in the system.
1. Westermeier R (2004). Isoelectric focusing. Methods Mol Biol 244, 225–232.