Isoelectric focusing

Isoelectric focusing (IEF) is a form of electrophoresis that sorts proteins by charge rather than by size. Every protein has an isoelectric point, or pI: the pH at which the positive and negative charges on its amino acid side chains exactly balance. At a pH below its pI a protein carries a net positive charge, and above it a net negative charge.

In IEF, proteins are placed in a gel strip or capillary holding a stable pH gradient, acidic at the positive electrode and basic at the negative. When a voltage is applied, each protein moves until it reaches the spot where the pH equals its pI. There it has no net charge and stops. Any protein that drifts away gains a charge and is pushed back, so each one gathers, or focuses, into a sharp band. The gradient was first formed by small buffering molecules called carrier ampholytes; today it is usually built into the gel as an immobilized pH gradient (IPG), which is more reproducible.

IEF can tell apart proteins that differ by a single charged amino acid or one added phosphate group. It is the first dimension of two-dimensional gel electrophoresis, followed by SDS-PAGE, and clinical labs use it to detect hemoglobin variants and the oligoclonal bands in cerebrospinal fluid that support a diagnosis of multiple sclerosis.