In this report, the optimization of a preparative electrophoretic method to fractionate secalins and hordeins is described. Separation was performed in preparative 7% polyacrylamide gels of 4 cm length at pH 3.1. The separation performance was tested using analytical electrophoresis at pH 3.1 and capillary electrophoresis (CE). Fractions of B- and C-hordeins were isolated in a single run from barley ethanol extract. γ- and ω-secalin fractions were isolated from rye ethanol extract. Resolution of preparative separation was maintained at a protein load of up to 30 mg in each run. Each secalin and hordein fraction showed several components of close mobility when analyzed by CE. Fractions from the preparative separation were pooled in such a way that no components from one pool were present in the others. These pooled fractions could be used as starting material for single polypeptide purification. Preparative electrophoresis at low pH allowed a simple separation of γ- and ω-secalins and B- and C-hordeins from crude material under non-denaturing conditions.Centro de Investigación y Desarrollo en Criotecnología de Alimentos