Cell fractionation and ultracentrifugation
summary notes
22 September 2022 14:20
Cell fractionation
- Cells are broken up and the different organelles they contain are
separated out
- Tissue is placed in cold, buffered solution of the same water
potential as the issue
Solution must be
1. Cold to reduce enzyme activity so organelles are not broken down
2. Same water potential as the tissue so that organelles do not burst
or shrink due to osmosis (isotonic)
3. Buffered so that pH does not change as this could change
structure of organelles or affect function of enzymes (ie denature
them)
Stage 1- Homogenation
- Cells broken up by homogeniser which is like a blender- breaks the
plasma membrane
- Releases organelles from cell
- Homogenate is resultant fluid that is filtered through a gauze to
remove any complete cells or debris
Stage 2- Ultracentrifugation
- Fragments in filtered homogenate are separated into centrifuge
machine
- Spins them at high speed to create centrifugal force
Process of ultracentrifugation:
- Tube of homogenate place in centrifuge at slow speed
- Heaviest organelles, the nucleus, forced to bottom of tube- form
sediment
- Fluid at the top, supernatant, is removed, leaving sediment
- Supernatant is transferred to another tube and spun in centrifuge
at faster speed than before (again so the heaviest organelles
forced to the bottom of the tube)
Mass
summary notes
22 September 2022 14:20
Cell fractionation
- Cells are broken up and the different organelles they contain are
separated out
- Tissue is placed in cold, buffered solution of the same water
potential as the issue
Solution must be
1. Cold to reduce enzyme activity so organelles are not broken down
2. Same water potential as the tissue so that organelles do not burst
or shrink due to osmosis (isotonic)
3. Buffered so that pH does not change as this could change
structure of organelles or affect function of enzymes (ie denature
them)
Stage 1- Homogenation
- Cells broken up by homogeniser which is like a blender- breaks the
plasma membrane
- Releases organelles from cell
- Homogenate is resultant fluid that is filtered through a gauze to
remove any complete cells or debris
Stage 2- Ultracentrifugation
- Fragments in filtered homogenate are separated into centrifuge
machine
- Spins them at high speed to create centrifugal force
Process of ultracentrifugation:
- Tube of homogenate place in centrifuge at slow speed
- Heaviest organelles, the nucleus, forced to bottom of tube- form
sediment
- Fluid at the top, supernatant, is removed, leaving sediment
- Supernatant is transferred to another tube and spun in centrifuge
at faster speed than before (again so the heaviest organelles
forced to the bottom of the tube)
Mass
