Fractionation of Cells

Fractionation of Cells - Subcellular Fractionation Ramnath...

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Subcellular Fractionation Ramnath Gowrishankar BIOL 3810-CTW Dr. Therese Poole Introduction:
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Gowrishankar 2 Subcellular fractionation is a biotechnological procedure that separates subcellular components using a series of increasing centrifugation speeds in numerous steps. The procedure begins by centrifuging cell lysate (broken cells) at a low centrifugation speed. At lower centrifugation speeds, only the densest cellular components will sediment (form a pellet) while 'lighter' components remain in the supernatant (non-pellet soluble phase). The supernatant can then be centrifuged at a higher speed to sediment the lighter cellular components still in solution. This incremental increase in centrifugation speeds is known as differential centrifugation. Differential centrifugation is a means by which subcellular components can be separated on the basis of their different densities. Thus, subcellular fractionation uses differential centrifugation to separate different subcellular components on the basis of their varying densities. The purpose of this lab is to separate nuclei from mitochondria. Since nuclei are bigger and denser than mitochondria, liver cells are broken and will be subject to a low centrifugation speed. This will theoretically sediment the nuclei while the lighter, less dense mitochondria remains in the supernatant. The supernatant from the low-speed centrifugation will then be centrifuged at a higher speed to sediment the smaller, less dense mitochondria. This second 'high-speed' centrifugation will produce a pellet that contains mitochondria and supernatant that contains lighter cell debris. To verify that pellets that contain the organelles they are supposed to
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Fractionation of Cells - Subcellular Fractionation Ramnath...

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