Cell Fractionation

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  • Created by: Jasmin
  • Created on: 17-09-13 20:10
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  • Cell Fractionation
    • Separating different parts and organelles of a cell
    • Differential centrifugation (spinning at high speed to produce centrifugal force)
    • Chop up fresh liver tissue in ice cold, isotonic buffer solution
      • Put chopped tissue in homogeniser to break open the cells which releases the organelles from the cell
        • Homogenate is filtered to remove any complex cells and large debris e.g. cell wall/membrane
          • Pour mixture in tubes and spin quickly in centrifuge
            • Faster the speed greater the force generated
            • Slower speed fragments collect at bottom, smaller remain at top suspended in liquid called supernatant liquid
            • Supernatant is poured in to a fresh tube, leaving sediment behind which contains nuclei
              • Larger fragment removed and supernatant remaining may be spun again at faster speed to produce sediment containing mitochondria and at even higher speed for other organelles
      • ICE COLD: reduce enzyme activity that might break down the organelles. Stop autolysis (splitting up)
      • ISOTONIC: prevent organelles bursting/shrinking as result of osmotic gain or loss of water. Has same water potential as original tissue. Stop osmotic effects
      • BUFFERED: to maintain a constant pH. Prevent damage to enzymes/proteins
    • Order of organelle fractionation (organelle, duration of centrifugation, speed of centrifugation/gravitational force)
      • Nuclei, 10min, 1000
      • Chloroplasts
      • Mitochondria, 10min, 3500
      • Lysosomes, 20mins, 16500
      • Endoplasmic Recticulum
      • Ribosomes, 60mins, 100000

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