Isoelectric focusing

Isoelectric focusing (IEF), 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. A protein that is in a pH region below its pI will be positively charged and so will migrate towards the cathode. As it migrates, however, the charge will decrease until the protein reaches the pH region that corresponds to its pI. At this point it has no net charge and so migration ceases. 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. The technique is capable of extremely high resolution with proteins differing by a single charge being fractionated into separate bands.

Molecules to be focused 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 molecules migrate through the pH gradient in the medium toward the "positive" end while positively charged molecules move toward the "negative" end. As a particle moves towards 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. 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 Immobilized pH gradient 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. [Stryer, Lubert: "Biochemie", page 50. Spektrum Akademischer Verlag, 1996 (German)] Isoelectric focusing is the first step in two-dimensional gel electrophoresis, in which proteins are first separated by their pI and then further separated by molecular weight through SDS-PAGE.

References

See also

* Alpha 1-antitrypsin where electrofocusing is used for diagnosis of the enzyme phenotype.

External links

* [http://instruct1.cit.cornell.edu/courses/biobm330/protlab/IEF.html Isoelectric Focusing Details]
* [http://microfluidics.stanford.edu/IEF_CE.htm Isoelectric Focusing Project] (read boxes on right for description of technique/apparatus.)