Differential Velocity Centrifugation

The several subcellular organelles are separated from one another on the behalf of their size in differential velocity centrifugation. A centrifuge  is used to produce  powerful  forces;  up  to  100 000  times  the  force  of  gravity  (g).  The homogenized sample is placed in an suitable centrifuge tube which is then loaded in the rotor of the centrifuge and subjected to centrifugation. For short periods at first relatively low g forces are used of time but then for longer time periods increasingly higher g forces are used. For instance, centrifugation at 600g for 3 min would pellet the nuclei, the largest organelles .The supernatant  from this step is removed to a fresh tube and then for 8 min centrifuged at 6000g to pellet out mitochondria,  peroxisomes  and, if present, chloroplasts or lysosomes.  Centrifugation  of this next supernatant  for 30 min at 40 000g will pellet out the plasma  membrane,  and fragments  of the Golgi  apparatus and endoplasmic reticulum.  A final  centrifugation  for 90 min at 100 000g  would result in a ribosomal  pellet and a supernatant  that is basically  free of specific  matter  and it  is considered  to be the true  soluble  cytosolic  fraction.

By differential velocity centrifugation Cell fractionation (a) format for subcellular fractionation of a tissue sample, (b) in the centrifuge tube appearance of a sample before and after centrifugation.

On the other hand,  by  differential  velocity  centrifugation  the  fractions  isolated  are  not entirely free of other subcellular  organelles usually and so may need to be purified  further.  A preparative centrifuge is used which has a rotor spinning in air at ambient pressure at low g forces for separations.  However, an ultracentrifuge is needed for separations at higher g forces. To reduce friction the chamber of the ultracentrifuge is kept in a high vacuum, and subsequent heating, which would otherwise take place between the spinning rotor and air.