Cell fractionation and ultracentrifugation Flashcards
What is needed to study one specific organelle?
a pure sample containing only the specific organelle being studied
What is cell fractionation?
the process of separating cell organelles from eachother
What are the three stages of cell fractionation?
homogenisation, filtration and ultracentrifugation
What is homogenisation?
the biological term used to describe the breaking up of cells.
What is the first step of homogenisation?
the sample of tissue (containing the cells to be broken up) must first be placed in a cold, isotonic buffer solution
In homogenisation, what are the three ways the solution must be?
Ice cold
Isotonic
Buffered
Why in homogenisation must the solution be ice cold?
to reduce the activity of enzymes that break down organelles
Why in homogenisation must the solution be isotonic?
to prevent water from moving into the organelles via osmosis, which would cause them to expand and eventually damage them
Why in homogenisation must the solution be buffered?
to prevent organelle proteins, including enzymes, from becoming denatured
What is the second step of homogenisation?
The tissue containing solution is then homogenised using a homogeniser.
What is a homogeniser?
A blender-like machine that grinds the cells up
What does the homogeniser cause?
This breaks the plasma membrane of the cells and releases the organelles into a solution called the homogenate
What happens in filtration?
The homogenate (containing the homogenised cells) is then filtered through a gauze
Why is the homogenate filtered through a gauze?
To separate out any large cell debris or tissue debris that were not broken up.
What is left after filtration?
A solution known as the filtrate that contains a mixture of organelles
What is the first step of ultracentrifugation?
The filtrate is placed into a tube and the tube is placed in a centrifuge
What is a centrifuge?
A machine that separates materials by spinning
What is the second step of ultracentrifugation?
The filtrate is spun first at a low speed
What does spinning the filtrate first at a low speed cause?
The largest, heaviest organelles (such as the nuclei) to settle at the bottom of the tube where they form a thick sediment known as the pellet
What happens to the rest of the organelles once the largest, heaviest ones have settled at the bottom?
They stay suspended in the solution above the pellet, known as the supernatant
What is the third step of ultracentrifugation?
The supernatant is dried off and placed into another tube which is spun at a higher speed. This causes the heavier organelles such as the mitochondria to settle at the bottom, forming a new pellet and leaving a new supernatant
The process of ultracentrifugation is repeated until…
all the different types of organelle present are separated out or just the desired organelle
Each new pellet formed contains a … organelle than the previous pellet
lighter
Give the usual order of mass for the organelles
nuclei, chloroplasts, mitochondria, lysosomes, endoplasmic reticulum, ribosomes
