Identification of tonoplast fractions resolved from plasma membrane by free-flow electrophoresis using filipin-labeling and antibody to the tonoplast ATPase

Abstract

Preparative free-flow electrophoresis has been employed in combination with density gradient centrifugation to prepare fractions enriched in either tonoplast or plasma membrane from dark grown seedlings of cress (roots), zucchini (hypocotyls), soybean (hypocotyls) and maize (coleoptiles). A polyclonal antibody to the 72,000 Mr subunit to the maize tonoplast ATPase was used to identify the tonoplast fractions from the free-flow electrophoresic separations and to show the absence of tonoplast contamination in plasma membranes derived from the same homogenates. These findings confirm the identity of the tonoplast fraction based on the presence of the proton translocating ATPase determined previously from sucrose gradient fractionation and inhibitor studies to be a tonoplast marker. Using staining with phosphotungstic acid at low pH, the plasma membrane fractions obtained after free-flow electrophoresis were shown to be > 90% plasma membrane-derived with little or no cross-contamination of plasma membrane vesicles in the tonoplast-containing fractions.