- Created by: babyblue18459
- Created on: 24-11-19 17:45
What is cell fractionation?
- The process of breaking up cells so their different organelles can be separated.
- It helps us to study the structure and function of cells and their organelles
- It is a two step process of homgenation and ultracentrifugation
- Tissue must be placed in a cold, isotonic, buffered solution
Why must the tissue be placed in a cold, isotonic,
- Cold - stops enzyme activity, which may break down organelles
- Isotonic - prevents organelles bursting/shrinking due to osmotic gain/loss of water. The isotonic solution has the same water potential as original tissue
- Buffered - keeps the pH constant
What are the steps for cell fractionation?
1. Tissue is cut into pieces and placed into a cold, isotonic and buffered solution
2. Homogenisation: Tissue is homogenised, for example, in a blender or homogeniser. This releases the organelles from the cell because the plasma membrane is broken down and organelles are released into solution.
3. Filtration: Homogenised suspension (homogenate) is filtered to remove any remaining whole cells or large debris e.g. cell wall or membrane. Organelles are smaller than debris, so pass through
4. Ultracentrifugation: The filtrate is placed in a small tube e.g. test tube, and is centrifuged at a low speed. The higher the speed of the spin, the greater the force.
5. The larger fragments collect at the bottom of the tube in a pellet. Smaller fragments remain suspended in a liquid called the supernatant
6. Supernatant is decanted and re-centrifuged at higher speeds for more force until the desired organelle separates into a pellet
Order of sedimentation as centrifuge speed increas
Pellet 1: Nucleus. Speed of centrifugation x gravity = 1000 with a spin of ten minutes
Pellet 2: Chlorplasts and mitochondria. Speed of centrifugation x gravity = 3500 with spin of ten minutes
Pellet 3: Endoplasmic reticulum, lysosomes. Speed of centrifugation x gravity = 16500 with spin of twenty minutes
Pellet 4: Ribosomes. Speed of centrifugation x gravity = 100000 with a spin of sixty minutes
What difficulties do scientists need to overcome w
- Organelles may be too small
- They might be inside the cell, and so hard to extract
- Once removed, the cell or organelles may not work