studying cells cell fractionation Flashcards
1.What is the purpose of cell fractionation?
To obtain a sample of a given organelle for study in the lab.
Give an overview of cell fractionation.
Cells are broken up (homogenisation) to release organelle, the debris is removed (filtration), and the organelles are separated using ultracentrifugation.
Describe how cells are homogenised.
The cell membrane is broken using a blender, releasing the organelles into the extraction solution.
. Describe and explain the composition of the extraction solution.
Ice-cold – Slows or stops enzyme activity to prevent digestion of organelles; Buffered – Maintains pH so that enzymes or proteins are not denatured; Same water potential as organelles– Prevents osmosis of water into/out of ORGANELLE so no bursting or shrinking of ORGANELLE (reject cell)
Explain why the homogenised cells need to be filtered.
To remove any cell debris or unbroken cells.
Describe and explain how organelles can be separated using ultracentrifugation.
The filtrate is placed in a test tube and spun at the lowest speed. The largest/heaviest/most dense organelles form a thick sediment at the bottom of the tube called a pellet. The supernatant, which is the fluid where all the other organelles are suspended, is poured off, and spun again at a higher speed. The next largest organelles form the pellet. This is repeated at progressively higher speeds until all the organelles are separated.
- Give the order that cell organelles will appear in the pellet at successively higher speeds.
Nucleus, mitochondria and chloroplasts, lysosomes, ribosomes
Explain how the organelles are separated by ultracetrifugation.
The organelles are separated on their mass, size and density. The largest/most dense/most massive are in the first pellet when a low spin speed was used.
