Isoelectric focusing (IEF) is an electrophoretic method for the separation of proteins according to their isoelectric points (pI). It is reproducible, sensitive and highly useful to resolve closely related proteins which may not be so well separated by other techniques.
Analytical IEF is carried out in a thin layer of polyacrylamide gel containing a large series of carrier ampholytes. When a potential difference is applied across the gel, the carrier ampholytes align themselves in order of increasing pI from the anode to the cathode, thus producing a uniform pH gradient across the gel. Under the influence of the electric field each protein migrates to the region in the gradient where the pH corresponds to its pI. The protein is electrically neutral at its pI and where it gets focused in the gel. After focusing, the separated components are detected by appropriate staining.