Monday, January 28, 2013

Isolation of Cell Organelles - Subcellular Fractionation

In this article, we are going to see how to isolate different organelles? First of all, I would like to ask, why is there a need to isolate organelles of the cell? I would argue that there is electron microscopy to see all the minute details of the cell. Then why to isolate different organelles?
The answer is, yes, definitely, the electron microscopy can be used for detailed visualization of the cell; however, it cannot determine the functions of the cell. Here comes the need to isolate the components of the cell - to study about its functions and hence, to do so, it is necessary to isolate them in intact form which can be used for biochemical studies. Hence, this can be accomplished using the technique, differential centrifugation followed by density-gradient centrifugation.

This method (differential centrifugation) is based on the principle that the cell components separate on the basis of the size and density. Following are the steps to separate cell components:
Diagrammatic Representation of Subcellular Fractionation
  • Disruption of plasma membrane/ Cell lysis: The first step in the process of differential centrifugation is the disruption of the plasma membrane or cell lysis in such a way that there is no harm to internal components of the cell i.e.; all the organelles remain intact. This can be done by several methods, like sonication (subjecting the cells to high-frequency sound); grinding in a homogenizer etc. This process is carried out in a buffer solution to maintain the osmotic environment to keep the components intact. This results in a solution containing broken cells, small fragments of plasma membrane, endoplasmic reticulum while it leaves the organelles of the cell (like mitochondria, nuclei, lysosomes etc.) intact. This solution is called a lysate or homogenate.

  • Ultracentrifugation:  The lysate is subjected to series of centrifugations which will fractionate into several components in an 'ultracentrifuge' which will rotate the samples at very high speeds. This will produce very high force and these forces will enable the components of the cell to settle down and form a pellet. This process of sedimentation will depend on the size and the density of the components with largest and heaviest components sedimenting first. The homogenate is first subjected to low speeds of around 800g for 10 minutes which will sediment unbroken cells and the largest cell organelle, nucleus. Thus, an enriched pellet of nucleus is obtained while supernatant (the remaining solution) contains other cellular components.
    The supernatant is further subjected to higher speeds (10,000-20,000g for 10 minutes) to sediment mitochondria, lysosomes, peroxisomes. The supernatant is again centrifuged to further high speeds (this time at 100,000 for 1 hour) resulting in fragments of plasma membrane and endoplasmic reticulum in the pellet.
    The final centrifugation step is done by further spinning it at very high speed which results in the ribosome sediments. The supernatant left is just cytosol.
The fractions which are obtained here, are enriched in corresponding organelle components, still, these fractions are not pure. So, to obtain pure components, further, density-gradient centrifugations have to be performed. In this process, the components are separated by sedimentation by a gradient of dense substance like sucrose. So, here, the sample is layered at the top of a gradient (sucrose). There is sedimentation of particles according to their sizes through gradient at different rates and discrete layers are formed. Thus, there is collection of each fraction which contains organelles of similar size (like mitochondria, lysosomes etc.)