Ultracentrifugation

A summary of the main points of ultracentrifugation.

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Process

  • Homogenate filtered
  • Low speed: remove unbroken cells
  • 600g: nuclei
  • supernatant spun 15000g: mitochondria, chloroplasts, lyosomes and peroxisomes
  • 100,000g: plasma membrane, endoplasmic reticulum, large polyribosomes.
  • Additional centrifugation: ribosomes, enzyme complex
  • 300,000g: Only the cytosol remains after centrifugation
  • This process means that at each stage of centrifugation, the next heaviest organelle is separated out.
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Definitions

  • Cellular homogenate is the liquid that contains the biological material. It must first be filtered to remove clumps.
  • The supernatant is the undeposited material left over after centrifugation.
  • Cytosol is the soluble aqueous portion of the cytoplasm.
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Information about the process

  • Vacuum conditions are used because this reduces heating caused by friction between air and the spinning rotor.
  • Tissue is placed into a cold isotonic buffered solution before cell fractionation occurs.
  • It is cold because this reduces enzyme activity which could break down organelles.
  • It is isotonic to prevent organelles bursting or shrinking as a result of osmotic water gain or loss.
  • It is buffered so that a constant pH is maintained.
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