Highly Purified Tonoplast Fractions by Preparative Free-Flow Electrophoresis
For the preparation of tonoplasts, the different approaches followed include isolation from homogenates by density gradient fractionation or from vacuoles prepared from protoplasts by osmotic lysis (Wagner, 1983). For our studies, we have used preparative free-flow electrophoresis, a procedure whereby highly purified fractions of both plasma membrane and of tonoplast are obtained from the same homogenate (Sandelius et al., 1986; Auderset et al., in press). In this technique, based on the methodology of Hannig and coworkers (Hannig and Heidrich, 1977), a mixture of components to be separated is introduced into a separation buffer moving perpendicular to the flux lines of an electric field (Fig. 1). Membranes bearing different electrical charge densities will migrate different distances across the separation chamber and thus may be resolved.
KeywordsPhosphotungstic Acid Plasma Membrane Vesicle Tonoplast Vesicle Glycerol Gradient Plasma Membrane Marker
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