Question

Describe the principle of ISO-electric focussing.

Answer 1

IEF, also known simply as electrofocusing, is a technique for separating charged molecules, usually proteins or peptides, on the basis of their isoelectric point (pI), i.e., the pH at which the molecule has no charge. IEF works because in an electric field molecules in a pH gradient will migrate towards their pI.
In most cases, a commercially available immobilized pH gradient (IPG) strip is used. The IPG strip consists of an acrylamide gel that contains wide pores to prevent a sieving effect based on protein mass, with a pH gradient. Various gradients are available, with wider gradients, such as pH 3-10 that are used for whole proteome analysis, and narrower ranges, such as pH 5-8 that are used for more specialist applications.
The sample is usually combined with carrier ampholytes to assist in migration. Ampholytes are a mixture of charged molecules with a range of pIs that matches the pI range of the IPG strip. The migration of the ampholytes encourages the sample molecules to move along the pH gradient. Ampholyte mixtures of a variety of pI ranges are commercially available.
After separation across the pH gradient, the sample is further separated (in 2D-PAGE) or analyzed (in the case of fractionation for mass spec–more on this below).