Cell Fractionation & Ultracentrifugation Flashcards
What is cell fractionation used for?
To isolate different organelles so they can be studied.
What are done to cells before fractionation?
They are broken open to release the contents and organelles.
In what type of solution are cells prepared in?
Cold, isotonic, buffered solution.
Why does the solution need to be cold?
To reduce enzyme activity which could damage the organelles.
Why does the solution need to be isotonic?
Organelles need to be at the same water potential as the solution to prevent osmosis which could cause the organelles to burst or shrivel.
Why does the solution need to be buffered?
The solution has a pH buffer to prevent damage to the organelles.
What are the 2 steps to cell fractionation?
Homogenisation and Ultracentrifugation
What is the first step to cell fractionation?
Homogenisation - the cells must be broken open (homogenised) using a blender. They’re blended in a cold, isotonic, buffered solution.
What is the second step to cell fractionation?
Filter the solution to remove large cell debris.
What is the third step to cell fractionation?
Ultracentrifugation - Separate organelles in the filtrate. The filtered solution is spun at high speeds in a centrifuge. This separates the organelles due to their densities. The most dense goes to the bottom to form a pellet.
What is differential centrifugation?
Doing it repeatedly at increasing speeds.
What is done each time in ultracentrifugation?
Process is repeated at faster speeds removing the supernatant each time leaving behind the pellets.
What is the supernatant?
The liquid at the top.
What is the order of organelles most dense to least dense?
Nuclei
Chloroplasts
Mitochondria
Lysosomes
ER
Ribosomes
What is the speed of centrifugation from nuclei to ribosomes?
1000
3500
N/A
10000
N/A
100,000
