cell fractionation + ultracentrifugation

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6 Terms

1
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what is cell fractionation?

  • process in which different parts and organelles of a cell are separated so they can be studied in detail

2
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name the 3 main stages of cell fractionation

  • homogenisation

  • filtration

  • ultracentrifugation

3
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what happens in homogenisation?

  • cells broken up using a homogeniser (blender)

    • breaks plasma membrane of cells + releases organelles into the homogenate (a solution)

    • carried out in a cold, isotonic, buffered solution

      • cold: slows enzyme activity

      • isotonic: prevents osmotic damage to organelles

      • buffered: maintains pH to avoid protein/enzyme denaturation

<ul><li><p>cells broken up using a homogeniser (blender)</p><ul><li><p>breaks plasma membrane of cells + releases organelles into the homogenate (a solution)</p></li><li><p>carried out in a cold, isotonic, buffered solution</p><ul><li><p><strong>cold: </strong>slows enzyme activity</p></li><li><p><strong>isotonic:</strong> prevents osmotic damage to organelles</p></li><li><p><strong>buffered:</strong> maintains pH to avoid protein/enzyme denaturation</p></li></ul></li></ul></li></ul><p></p>
4
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what happens in filtration?

  • homogenate is filtered through a gauze to remove large debris

  • organelles remain in the filtrate (filtered solution)

5
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what happens in ultracentrifugation?

  • filtrate placed into a tube → tube placed in a centrifuge (machine which separates materials by spinning)

  • filtrate spun in centrifuge at increasing speeds

    • heaviest organelles form a pellet at the bottom while the rest stay suspended in the solution above the pellet } solution is called ‘supernatant’

  • supernatant is re-spun at higher speeds to isolate lighter organelles

  • process repeated at increasing speeds until all different types of organelle present are separated out

<ul><li><p>filtrate placed into a tube → tube placed in a centrifuge (machine which separates materials by spinning)</p></li><li><p>filtrate spun in centrifuge at increasing speeds</p><ul><li><p>heaviest organelles form a pellet at the bottom while the rest stay suspended in the solution above the pellet } solution is called ‘supernatant’</p></li></ul></li><li><p>supernatant is re-spun at higher speeds to isolate lighter organelles</p></li><li><p>process repeated at increasing speeds until all different types of organelle present are separated out</p></li></ul><p></p>
6
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order the weight of organelles from heaviest to lightest

  • nucleus

  • chloroplasts

  • mitochondria

  • lysosomes

  • endoplasmic reticulum

  • ribosomes