Conventional carrier ampholyte‐isoelectric focusing is unsuitable for separating wool and hair proteins. The inability to form stable narrow pH gradients at acidic pH values does not allow good resolution of the many acidic wool and hair proteins. To evaluate the usefulness of immobilised pH gradient‐isoelectric focusing (IPG‐IEF) for wool proteins, S‐amidomethyl wool proteins were analysed using IPG‐IEF. Over twenty bands were visible on a pH 4–7 IPG‐IEF gel. IPG‐IEF was combined with sodium dodecyl sulphate‐polyacrylamide gel electrophoresis (SDS‐PAGE) to give a two dimensional separation, and over 50 protein spots were observed. IPG‐IEF appears to be ideally suited to the separation of wool proteins. The resolution of two‐dimensional electrophoresis using IPG‐IEF was greater than previously obtained using acid or alkaline PAGE in the first dimension. Good reproducibility of spot position was observed.