Isotachophoresis on Polyacrylamide Gel

Abstract

Abstract Isotachophoresis, using Ampholine spacer ions, was applied to the fractionation of two multicomponent protein systems (serum and a urinary preparation tract with Hunter Factor activity) using polyacrylamide gel as a supporting medium. These studies were designed to determine whether isotachophoresis could provide higher load capacity than obtained in conventional electrophoresis or isoelectric focusing on polyacrylamide gel without loss of resolution. The effect of the Ampholine pI range, the relative and absolute concentrations of protein sample and Ampholine, of the ionic strength of the gel buffer, and of the stacking limits obtained in various gel buffers was investigated. It was found that Ampholine components can act as spacers, giving rise to bands in what otherwise would be a continuum of stacked protein zones. Loads greater by 1 to 2 orders of magnitude than in conventional polyacrylamide gel electrophoresis were applicable. The resotion obtained was greatly inferior to that in PAGE, suggesting that Ampholine does not provide a sufficient distribution of constituent mobilities under the employed conditions. Isotachophoresis was applied at the preparative scale in two-stage fractionations designed to resolve multicomponent systems (serum).