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3.3 outline the role of centrifugation in removing cell debris and sedimenting cell organelles, such as chloroplasts
outline the role of centrifugation in removing cell debris and sedimenting cell organelles, such as chloroplasts
- Spinning in an ultra-centrifuge (100 000 rpm or 500 000 times gravity) separates the fractions or parts of the cell. Cell components move to the bottom of the centrifuge at a rate determined by their size and density, the largest and heaviest falling to the bottom first. If a suspension of broken cells (called an homogenate or lysate) is spun at different rates, the following fractions can be separated:
- low speed – unbroken cells and nuclei can be removed in a pellet (sediment) at the bottom of the tube leaving the rest of the cell components in solution above (the supernatant).
- centrifuging the supernatant at higher speed sediments the chloroplasts, mitochondria and lysosomes.
- re-centrifuging the supernatant at an even higher speed sediments the plasma membrane and the endoplasmic reticulum.
- a fourth centrifugation at a still higher speed sediments the ribosomes, leaving only the cytosol (cytoplasm) in the supernatant.
- To produce a pure chloroplast preparation density gradient centrifugation is preferred. To make a density gradient, different concentrations of sucrose solutions are used in turn, layered on top of one another in a centrifuge tube. The most concentrated solution is at the bottom and the least concentrated is on the top. The homogenate is placed carefully on top of the layers and spun in a centrifuge. Organelles of different sizes settle out as discrete bands through the gradient at the interface between sucrose solutions, depending on their size and density.