density-gradient centrifugation

A technique for separating and isolating pure samples of cell organelles, i.e. cell fractionation. The cells are first broken up in a homogenizer to release the contents and the homogenate is filtered to remove cell wall fragments. It is then poured on top of prepared salt or sucrose solutions of different concentrations that have been layered according to their density in a glass tube. Provided an appropriate centrifugal force is applied, the cell inclusions will band together at the regions in the density gradient that correspond to their own density and can then be separated off by various methods. Another method of isolating cell organelles, differential centrifugation, involves the filtered homogenate being centrifuged and decanted several times in sequence, increasing the time and speed of centrifugation at each successive stage. Nuclei, membranes, chloroplasts, mitochondria, vesicles, endoplasmic reticulum, and ribosomes, are collected in that order.