3.9 - Cell fractionation

Question 1

Process of ultracentrifugation

Answer 1

1. The cell fragments are placed in a centrifuge tube and spun at a low speed.
2. This results in the heaviest organelles, such as nuclei, forming a pellet at the bottom of the tube.
3. The lighter organelles remain suspended in the supernatant.
4. The supernatant is then transferred to a new tube and centrifuged at a higher speed.
5. This leads to the next heaviest set of organelles, typically mitochondria, settling into a pellet.
6. Steps 4 to 5 are repeated, increasing the speed each time to separate the remaining organelles until all organelles have been separated into distinct layers.

Question 2

What is a supernatant?

Answer 2

The liquid remaining above the pellet, which contains the lighter organelles.

Question 3

What is a pellet?

Answer 3

sediment at the bottom of the tube, containing the heavier organelles.

Question 4

Cell fractionation method

Answer 4

1. prepare the tissue sample in a;

• Ice-cold- This slows down enzyme activity that might otherwise break down organelles.
• Isotonic- It ensures that the water potential inside and outside the organelles is the same, preventing damage through water movement.
• Buffered - Keeping the pH constant is crucial to prevent denaturation of proteins and enzymes.

2. homogenise it;

physically breaking open the cells. This disrupts the plasma membrane, which allows the organelles to be released into the solution. (a blender)

3. the mixture is then filtered to remove larger pieces of cell debris and any remaining tissue fragments.The filtration is typically done through a gauze, which allows smaller organelles to pass through while retaining larger debris.
4. Ultracentrifugation;

separate organelles based on their density. By spinning the filtered solution at various speeds, organelles are separated into layers according to their mass

Question 5

What is cell fractionation?

Answer 5

a laboratory method used to isolate different components of the cell, specifically the organelles, so they can be studied in detail, such as under an electron microscope