Cell Fractionation Flashcards
What are the steps of cell fractionation?
- Homogenisation
- Filtration
- Ultracentrifugation
Describe homogenisation
- break down cells to release organelles by vibrating cells or grinding cells in a blender
- conditions of solution: ice cold, isotonic, buffered
What are the 2 methods to break plasma membrane and release organelles?
- Vibrating cells
- Grinding cells in a blender
What are the 3 conditions of the homogenised solution?
- Ice cold
- Isotonic
- Buffered
Why should the homogenised solution be ice cold?
Slow down enzyme activity to prevent denaturing
Why should homogenised solution be isotonic?
Water potential of the solution should be the same as the cells that have been broken down to prevent osmosis and therefore damage
Why should the homogenised solution be buffered?
Ensure pH stays constant so proteins are not denatured
Describe the process of filtration
- homogenised solution filtered through a gauze to remove tissue and cell debris
- organelles are much smaller than debris so aren’t filtered out
- leaves filtrate containing mixture of organelles
Describe the process of ultracentrifugation
- filtrate poured into tube and put into centrifuge and spun at low speed
- supernatant drained off and poured into another tube and spun at higher speed
- process repeated at increasing speeds until all organelles are separated
What are pellets?
The heaviest organelle settled at the bottom of the tube
What is the mass order from pellet 1-4?
- Nuclei
- Mitochondria and chloroplast
- Lysosomes
- Endoplasmic reticulum and ribosomes