Differential centrifugation

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Differential centrifugation is a procedure in which a homogenate is subjected to repeated centrifugations each time increasing the centrifugal force. Separation is based predominantly on particle mass and size with larger and heavier particles pelleting at lower centrifugal fields. As an example, unbroken whole cells will pellet at low speeds and short intervals such as 1,000 x g for 5 minutes. Whereas smaller cell fragments and organelles require more force and greater times to pellet. In general, one can enrich for whole cells, nuclei, mitochondria and lysosomes, microsomes (vessicles of Golgi and endoplasmic reticulum) and/or cytosol (particle free solution of the cytoplasm), respectively, by exposing a homogenate and subsequent supernatants to sequentially greater centrifugal fields.

Density centrifugation uses a dense solution or density gradient to separate particles based on their individual densities or mass/size ratio. A solution is prepared such that a gradient of densities is available for particles to pass through or float upon. This density gradient may be continuous or prepared in a stepped manner such as the layers in a parfait. For instance, sucrose is often used to prepare density gradients and one can carefully float a solution of 40% sucrose onto a layer of 45% sucrose and add further less dense layers above. The homogenate, prepared in a dilute buffer and centrifuged briefly to remove tissue and unbroken cells, is then layered on top. After centrifugation typically for an hour at about 100,000 x g, one can observe disks of cellular components residing at the change in density from one layer to the next. By carefully adjusting the layer densities to match the cell type, one can enrich nuclear, mitochondrial, microsomal, and plasma membrane vessicle fractions. In another common practice, cesium chloride is mixed with a buffer and a continuous density gradient forms during the centrifugation. Nucleic acids such as plasmid and genomic DNA as well as RNA appear as floating clouds in this gradient after centrifugation and can be extracted for further purposes. This procedure is also know as "equilibrium density centrifugation".

[edit] Ultracentrifugation

An ultracentrifuge consists of a refrigerated, evacuated chamber containing a rotor which is driven by an electrical motor capable of high speed rotation. Samples are placed in tubes within or attached to the rotor. Rotational speed may reach around 70,000 rpm, creating centrifugal speed forces of 500,000g. This force causes sedimentation of macromolecules, and can even cause non-uniform distributions of small molecules. Sedimentation depends on mass, shape, and partial specific volume of a macromolecules, as well as solvent density, rotor size and rate of rotation. The sedimentation velocity can be monitored during the experiment to calculate molecular weight. Values of sedimentation coefficient (S) can be calculated. Large values of S (faster sedimentation rate) correspond to larger molecular weight. Dense particle sediments more rapidly. Elongated proteins have larger frictional coefficients, and sediment more slowly.