Differential centrifugation

Differential centrifugation is a common procedure in microbiology and cytology used to separate certain organelles from whole cells for further analysis of specific parts of cells. In the process, a tissue sample is first homogenised to break the cell membranes and mix up the cell contents. The homogenate is then subjected to repeated centrifugations, each time removing the pellet and increasing the centrifugal force. Finally, purification may be done through equilibrium sedimentation, and the desired layer is extracted for further analysis.

Separation is based on size and density, with larger and denser particles pelleting at lower centrifugal forces. As an example, unbroken whole cells will pellet at low speeds and short intervals such as 1,000g for 5 minutes. Smaller cell fragments and organelles remain in the supernatant and require more force and greater times to pellet. In general, one can enrich for the following cell components, in the separating order in actual application:

  • Whole cells and nuclei;
  • Mitochondria, lysosomes and peroxisomes;
  • Microsomes (vesicles of disrupted endoplasmic reticulum); and
  • Ribosomes and cytosol.

Sample preparation

Before differential centrifugation can be carried out to separate different portions of a cell from one another, the tissue sample must first be homogenised. In this process, a blender, usually a piece of porous porcelain of the same shape and dimension as the container, is used. The container is, in most cases, a glass boiling tube.

The tissue sample is first crushed and a buffer solution is added to it, forming a liquid suspension of crushed tissue sample. The buffer solution is a dense, inert, aqueous solution which is designed to suspend the sample in a liquid medium without damaging it through chemical reactions or osmosis. In most cases, the solution used is sucrose solution; in certain cases brine will be used. Then, the blender, connected to a high-speed rotor, is inserted into the container holding the sample, pressing the crushed sample against the wall of the container.

With the rotator turned on, the tissue sample is ground by the porcelain pores and the container wall into tiny fragments. This grinding process will break the cell membranes of the sample's cells, leaving individual organelles suspended in the solution. This process is called homogenization. A portion of cells will remain intact after grinding and some organelles will be damaged, and these will be catered for in the later stages of centrifugation.

Ultracentrifugation

The homogenised sample is now ready for centrifugation in an ultracentrifuge. 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 up to 100,000 rpm for floor model, 150,000 rpm for bench-top model (Beckman Optima Max-XP), creating centrifugal speed forces of 800,000g to 1,000,000g. This force causes sedimentation of macromolecules, and can even cause non-uniform distributions of small molecules.

Since different fragments of a cell have different sizes and densities, each fragment will settle into a pellet with different minimum centrifugal forces. Thus, separation of the sample into different layers can be done by first centrifuging the original homogenate under weak forces, removing the pellet, then exposing the subsequent supernatants to sequentially greater centrifugal fields. Each time a portion of different density is sedimented to the bottom of the container and extracted, and repeated application produces a rank of layers which includes different parts of the original sample. Additional steps can be taken to further refine each of the obtained pellets.

Sedimentation depends on mass, shape, and partial specific volume of a macromolecule, 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 to ensure accuracy.

Equilibrium (isopycnic) sedimentation

Equilibrium sedimentation uses a gradient of a solution such as cesium chloride or sucrose to separate particles based on their individual densities (mass/volume). It is used as a purifying process for differential centrifugation. A solution is prepared with the densest portion of the gradient at the bottom. Particles to be separated are then added to the gradient and centrifuged. Each particle proceeds (either up or down) until it reaches an environment of comparable density. Such a density gradient may be continuous or prepared in a stepped manner. For instance, when using sucrose to prepare density gradients, 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 for specific cellular components. Caesium chloride allows for greater precision in separating particles of similar density. In fact, with a caesium chloride gradient, DNA particles that have incorporated heavy isotopes (13C or 15N for example) can be separated from DNA particles without heavy isotopes.


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