Organelle isolation

Abstract Subcellular fractionation has, as its general principle, the application of three consecutive procedures. The first, homogenization, results in the abolition of cellular integrity. Depending on the forces involved, individual compartments or organelles may also no longer remain intact. The second step entails imposing a new system of order among the randomly dispersed organelles in the homogenate. This is usually achieved through separations based on size (gel filtration), mass (differential or rate zonal centrifugations), density (isopycnic centrifugation), or surface charge (electrophoresis, phase-partitioning). Finally, the subcellular units or fractions obtained have to be checked for their homogeneity. For this, electron microscopical (negatively stained or thin sectioned samples), chemical (enzyme tests), and immunological (Western blotting) methods can be employed.