Preparative fractionation of gliadins by electrophoresis at pH 3.1 (A-PAGE)

Purification of gliadin subclasses has been difficult since they share many biochemical and physicochemical properties. In this report, the optimizati on of a preparative electrophoretic method to fractionate gliadins is descr ibed. Separation was performed in preparative 7% polyacrylamide gels at pH 3.1. The separation performance was tested using analytical electrophoresis at pH 3.1 and capillary electrophoresis. Preparative gels of different len gths were employed. Using 5-cm preparative gels, several fractions of alpha -, beta-, and gamma-gliadins and fast-mobility and slow-mobility omega-glia dins were collected in 40 h of separation. Resolution was maintained at a p rotein load of up to 30 mg in each run. The highest efficiency of recovery was achieved using aluminum lactate as the collecting buffer. Fractionation with 10 cm in length gels improved resolution but increased operation time s. Gels of 2 cm in length did not separate alpha/beta and gamma-gliadins ef ficiently but were useful to separate the two main fractions of omega-gliad ins in shorter times. In conclusion, preparative electrophoresis at low pH allowed the separation of alpha-, beta-, gamma-, and omega-gliadin fraction s from crude material under nondenaturing conditions.