Ultracentrifugation Flashcards
What is cell fractionation?
It is a process where cells are broken up and their different organelles are separated and collected based on their size. This is done using gravity and three different processes.
What are the three processes used in cell fractionation?
They are homogenisation, filtration and ultracentrifugation.
What are the properties needed for the solution?
The solution needs to be ice cold to reduce enzyme activity which prevents organelles from breaking down. It also needs to be isotonic (which means having the same water potential as the tissue), preventing organelles from bursting or shrivelling due to osmosis. Finally, the solution must be buffered to maintain a constant pH.
What is homogenisation?
It involves breaking up the tissue to open the cell membranes and release the organelles inside.
What is filtration?
The homogenate (homogenised solution) is filtered in order to remove large debris and cells that didn’t break open. This is because they would sink to the bottom of the test tube and distrupt the centrifuging.
What is ultracentrifugation?
- At a slow speed, the test tube is spun, which makes the larger organelles form a pellet at the bottom. This can now be collected.
- At this point, the smaller organelles are still at the top in the liquid called the supernatant.
- The supernatant is respun at higher and higher speeds so the smaller organelles can form their own pellets. Each time a pellet is formed, it is collected so the next largest organelle can take its turn.
What order are the fractionalised organelles collected?
The heaviest organelles form pellets and are collected first. Therefore, the nuclei is collected first, then the chloroplasts, mitochondria, lysosomes, endoplasmic reticulum and finally ribosomes.
