Fig. 1.

Centrifugation for subcellular fractionation. Differential centrifugation is usually the first step after cell lysis in subcellular fractionation protocols and is often followed by density gradient centrifugation. The fragments of the cell wall, unlysed cells, and the nuclei can be sedimented at low speed (3,000–5,000 × g). The pellet can be subjected to density gradient centrifugation for further purification of the nuclei. The postnuclear supernatant, containing all other organelles and the plasma membrane vesicles, is usually subjected to density centrifugation for further fractionation. The postnuclear supernatant can also be subjected to a second round of differential centrifugation at higher g force (20,000–30,000 × g). The mitochondria and peroxisomes are then further purified from the pellet using density gradient centrifugation, and the vacuoles, smooth ER (SER), and plasma membrane (PM) vesicles can be purified from the supernatant. Alternatively, the supernatant can be subjected to ultracentrifugation to sediment light organelles and organellar vesicles and separate these from the cytosol and lipid particles that remain in the supernatant. We emphasize that the figure is an oversimplification because of limitations to the designation of organelle names to crude differential fractions. RER, rough ER.