Preparative isotachophoresis in a flat bed of granulated gel. I. Principles and procedures, comparison with isoelectric focusing and application to the isolation of a low pI/high mobility form of cat liver cytosolic glutathione S‐transferase

Abstract A method is reported for the separation and partial purification of proteins on a preparative scale (around 100 mg or more total protein per separation run), using the isotachophoretic principle. The technique was carried out using a granulated gel (Ultrodex) as supporting medium, with conventional flat‐bed electrophoresis equipment. This method was applied to the separation of multiple forms of glutathione S‐transferase, derived from the soluble cytosolic fraction of cat liver homogenate. Of particular interest in the present work was the isolation of the highest mobility form of the enzyme, which also functions as a cytosolic ligand‐binding protein for bilirubin. The advantages and disadvantages of isotachophoresis are compred with those of isoelectric focusing, using a simliar apparative set‐up. The latter had the primary drawback that some forms of the enzyme, particularly those with most acidic or alkaline isoelectric points, were, to a certain extent, irreversibly inactivated following focusing, whereas isotachophoresis yielded considerably higher volume activities and resulted in less inactivation during separation. The loss in activity of some forms of the enzyme was particularly marked in isoelectric focusing in thinner gels with agarose as supporting phase. Isotachophoresis has become associated with quite complicated instrumental requirements. The simple form of this technique reported in the present work should provide a useful addition to the spectrum of methods available for protein separation and purification. The major limitation of the flat‐bed configuration is that the actual resolution obtained cannot be fully exploited owing to the technical difficulties associated with sectioning the flat‐gel.