Purification of yeast membranes and organelles by sucrose density gradient centrifugation

Many experiments require isolation and purification of membranes and organelles from a cell-free lysate. A combination of differential and sucrose density gradient centrifugation provides adequate separation of most yeast organelles in a single experiment. Yeast cells are converted to spheroplasts and gently lysed under conditions that preserve the integrity of organelles. The total lysate is subjected to differential centrifugation and the resulting membrane pellets are fractionated on density gradients. The method is based on the fact that different membranes contain different ratios of lipid to protein, and thus exhibit different density, allowing them to migrate through the gradient until they reach isopycnic position. The fractionated gradients are analyzed by Western blotting with antibodies that recognize marker proteins specific for individual organelles.