Cell Fractionation Methods

Written by john brennan
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Cell Fractionation Methods
Differential centrifugation is a key part of cell fractionation. (Thinkstock Images/Comstock/Getty Images)

Biologists often need to purify specific cell components or proteins from tissue samples as part of an experiment. The process by which they separate cells into their components is called cell fractionation. There are several steps to this process

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Homogenisation

First the cells must be broken open. A variety of different methods are available; the method chosen depends on the type of experiment and the type of sample (bacterial culture or mammalian tissue sample, for example) Detergents like SDS or Triton X disrupt the cell membrane so the contents can flow out. Subjecting the cells to ultrasound waves or sonicating them will also break them open, as will agitation in the presence of metal or glass beads. Blenders may work with tissue samples but will not work with bacteria or other microorganisms.

Filtration

Cells are always homogenised in a buffered solution, which helps to stabilise pH and thereby prevent damage to cell contents during extraction. Occasionally the resulting homogenate must be filtered to remove connective tissue or other undesirable elements. Depending on the nature of the contaminant, this can be as simple as pouring the homogenate through gauze and collecting the filtrate on the other side. At all times following homogenisation, the homogenate should be kept cold to minimise the activity of proteases, enzymes that chop up proteins. Sometimes protease inhibitors are added as well.

Centrifugation

Next, the homogenate is transferred to a centrifuge tube and placed in a centrifuge. This device contains a rotor that spins at high speed, flinging the contents of each centrifuge outwards -- in much the same way that driving left around a sharp curve flings you towards the passenger-side door. Higher-density components of the homogenate sediment sooner and at lower speeds than lower-density components, so the components of the homogenate can be separated and collected on the basis of density.

Ammonium Sulfate Precipitation

Sometimes biologists need to collect specific organelles from the homogenate for further study. If so, they will use repeated centrifugation to isolate the fraction that contains the organelle they need. If, on the other hand, they are trying to isolate a specific protein, they may use a technique called ammonium sulphate precipitation. By adding ammonium sulphate salt, they increase the ionic strength of the solution, causing some proteins to precipitate following centrifugation. This process reduces the protein concentration in the homogenate and will make it easier to purify the protein of interest later on.

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