Cell Fractionation
- Created by: Jasmin
- Created on: 17-09-13 20:10
View mindmap
- 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
- Pour mixture in tubes and spin quickly in centrifuge
- Homogenate is filtered to remove any complex cells and large debris e.g. cell wall/membrane
- 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
- Put chopped tissue in homogeniser to break open the cells which releases the organelles from the cell
- 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
Comments
No comments have yet been made