Cell fractionation + Ultracentrifugation Flashcards
How can you study the structure and function of various organelles?
To study the structure and function of various organelles that make up cells, large numbers of isolated organelles need to be collected
What is cell fractionation?
Cell fractionation is the process where cells are broken up and the different organelles they contain are separated out.
What occurs before cell fractionation can begin?
Before cell fractionation begins the tissue has to be placed in a cold, isotonic buffered solution of the same water potential as the tissue
Why is the solution that tissues are placed, cold?
The solution is cold to reduce enzyme activity that might break down the organelles
Why is the solution that the tissues are placed, isotonic?
The solution is isotonic to keep the water potential the same. This prevents organelles bursting or shrinking as a result of osmotic gain or loss of water
Why is the solution that the tissues are placed, buffered?
The solution is buffered so that the pH does not fluctuate. Any change in pH could alter the structure of the organelles or affect the functioning of enzymes
How many stages are there in cell fractionation? What are the names of these stages?
There are two stages of cell fractionation: homogenisation and ultracentrifugation
What is the first stage of cell fractionation?
The first stage of cell fractionation is homogenisation
What occurs during homogenisation?
During homogenisation, cells are broken up by a homogeniser (blender) which releases the organelles from the cell. This results in a fluid known as homogenate and it is then filtered to remove any complete cells and large pieces of debris
What is the resultant fluid called after being placed in the homogeniser?
The resultant fluid is called homogenate
What occurs to the homogenate before it moves onto the next stage of cell fractionation?
The homogenate is then filtered to remove any complete cells and large pieces of debris
What is ultracentrifugation?
Ultracentrifugation is the process by which the fragments in the filtered homogenate are separated in a machine called a centrifuge
How does the centrifuge work?
The centrifuge spins tubes of homogenate at different speeds to create a centrifugal force and allowing organelles to separate according to their densities
What are the steps of ultracentrifugation?
- The tubes of homogenate are spun at a low speed an the process is repeated at increasingly faster speeds
- Each time the supernatant (liquid) is removed, pellets of organelles are left
- The supernatant is then spun again to remove the next pellet of organelles until there are no organelles left to remove
What does the centrifugal forces cause?
The centrifugal forces cause pellets of the most dense organelles to form at the bottom
