Purification techniques of subcellular compartments for analytical and preparative purposes.
Résumé
In the following protocols, broken cells are the starting material of all downstream purifications of functional organelles or intact subcellular membranes. The choice of the breakage method has direct and deep repercussions on the quality of subsequent steps. Breaking vegetative amoebae by shear stress with a steel ball cell cracker preserves the integrity of subcellular organelles and in particular that of lysosomes, the rupture of which is very deleterious to further purifications. In this chapter, we propose purification schemes for plasma membrane, nuclei, mitochondria, and endocytic compartments. Plasma membranes are purified without any cell coating by partition between aqueous polymer phases. Nuclei and mitochondria are purified by differential centrifugations in adequate buffer conditions. Endosomes are magnetically isolated after feeding the cells with colloidal iron dextran and phagosomes by flotation on a sucrose gradient after feeding amoebae with latex beads. As analytical approaches, we propose procedures to label the plasma membrane and the endo-lysosomal compartments by biotinylation and to separate early and late compartments on a Percoll gradient.