By concentrating the enzymes of particular metabolic pathways into distinct and small regions of its interior, the cell can increase the rate of particular biochemical pathways by several orders of magnitude. Isoelectric focusing is the only step in preparative one-dimensional gel electrophoresis.Īccording to some opinions, living eukaryotic cells perform isoelectric focusing of proteins in their interior to overcome a limitation of the rate of metabolic reaction by diffusion of enzymes and their reactants, and to regulate the rate of particular biochemical processes. Isoelectric focusing is the first step in two-dimensional gel electrophoresis, in which proteins are first separated by their pI value and then further separated by molecular weight through SDS-PAGE. Isoelectric focusing can resolve proteins that differ in pI value by as little as 0.01. Gels with large pores are usually used in this process to eliminate any 'sieving' effects, or artifacts in the pI caused by differing migration rates for proteins of differing sizes. Proteins are introduced into an Immobilized pH gradient gel composed of polyacrylamide, starch, or agarose where a pH gradient has been established. 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. 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. 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. As a particle moves toward the pole opposite of its charge it moves through the changing pH gradient until it reaches a point in which the pH of that molecules isoelectric point is reached. Negatively charged molecules migrate through the pH gradient in the medium toward the 'positive' end while positively charged molecules move toward the 'negative' end. An electric current is passed through the medium, creating a 'positive' anode and 'negative' cathode end. Molecules to be focused are distributed over a medium that has a pH gradient (usually created by aliphatic ampholytes). The technique is capable of extremely high resolution with proteins differing by a single charge being fractionated into separate bands. As a result, the proteins become focused into sharp stationary bands with each protein positioned at a point in the pH gradient corresponding to its pI.

At this point it has no net charge and so migration ceases (as there is no electrical attraction toward either electrode). As it migrates through a gradient of increasing pH, however, the protein's overall charge will decrease until the protein reaches the pH region that corresponds to its pI. An immobiline is a weak acid or base defined by its pK value.Ī protein that is in a pH region below its isoelectric point (pI) will be positively charged and so will migrate toward the cathode (negatively charged electrode). The immobilized pH gradient is obtained by the continuous change in the ratio of immobilines. IPGs are the acrylamide gel matrix co-polymerized with the pH gradient, which result in completely stable gradients except the most alkaline (>12) pH values. IEF involves adding an ampholyte solution into immobilized pH gradient (IPG) gels.

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. Isoelectric focusing ( IEF), also known as electrofocusing, is a technique for separating different molecules by differences in their isoelectric point (pI).

Electrophoresis cathode charge