Isoelectric focusing

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Isoelectric focusing, also known as electrofocusing, is a technique for separating different molecules by their electric charge differences. It is a type of zone electrophoresis, usually performed in a gel, that takes advantage of the fact that a molecule's charge changes with the pH of its surroundings.

Molecules are distributed over a medium that has a pH gradient (usually created by aliphatic ampholytes). An electric current is passed through the medium, creating a "positive" anode and "negative" cathode end. Negatively charged particles migrate through the pH gradient toward the "positive" end while positively charged particles move toward the "negative" end. As a particle moves towards the pole opposite of it's charge it moves through the changing pH gradient until it reaches a point in which the pH of that molecules isoelectric point is reached. 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 initially 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. Proteins are introduced into a gel composed of polyacrylamide, starch, or agarose where a pH gradient has been established. 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. Isoelectric focusing can resolve proteins that differ in pI value by as little as 0.01.^[1] Isoelectric focusing is the first step in two-dimensional gel electrophoresis, in which proteins are subsequently separated by molecular weight through SDS-PAGE.