Isoelectric focusing

Isoelectric focusing is an analytical purification technique that separates proteins, electrophoretically, according to their acidic or basic intrinsic charges i.e. their isoelectric point.

A polyacrylamide gel apparatus containing a pH gradient is used. The pH gradient is established by the subjection of a buffer-like mixture of many polyampholytes, each with a different isoelectric point, to electrophoresis. The protein sample is then loaded and a suitable voltage is applied to the electric field. The protein migrates to its characteristic isoelectric pH forming a band and, once reached, its electrophoretic mobility becomes zero; movement in the positive or negative direction is inhibited.

Isoelectric focusing can distinguish between proteins that differ by as little as one net charge i.e. a pI value of 0.01.