Cell Fractionantion/Differential Centrifugation Flashcards
What are the three main steps involved?
Homogenisation, (Filtration), Ultracentrifugation
What is this technique used for?
To separate undamaged, intact organelles from the rest of the cell so they can be studied under a microscope.
What is the first step?
Chop up the sample in an ice cold, isotonic, buffer (solution).
In the first stage, why is an ice cold solution used?
To reduce the activity of enzymes that break down organelles.
In the first stage, why is an isotonic solution used?
(A solution which has the same concentration of chemicals as the cells being broken down) To prevent the shrinking or bursting of organelles via osmosis.
In the first stage, why is a buffer used?
To prevent a pH change which would affect the enzymes.
What is the second stage?
Put the chopped tissue into a blender or HOMOGENISER (or you could vibrate the cells) which breaks open the cells by breaking the plasma membrane and releasing the organelles into solution. This is homogenisation.
What is the third stage?
Filter the mixture using a gauze to remove any debris (eg. insoluble tissue - connective tissue from the organelles) keeping it cool throughout.
What is the fourth stage?
Ultracentrifugation - The cell fragments are poured into a tube which is then spun at a low speed in a CENTRIFUGE.
In the fourth stage, what is formed after the mixture has been spun?
The denser organelles get spun to the bottom of the tube where they form a ‘pellet’ called the sediment. A liquid layer is formed on top this is the supernatant. This is where the rest of the organelles stay suspended.
What is the fifth stage?
The supernatant is poured into a fresh tube leaving the sediment behind. This is then spun at a faster speed to produce a sediment containing the next most dense organelle.
What is the sixth stage?
The supenanant is then removed and the process is repeated again and again at higher and higher speeds until all of the organelles are separated out.
What is the order in which the organelles form pellets?
From heaviest to lightest - Nuclei (at a speed of 1000), Chloroplasts (in plant cells), Mitchondria (3,500), Lysosomes (16,500), Endoplasmic reticulum, Ribosomes (100,000)
What is another way to separate organelles?
Density Gradient Centrifugation - sucrose is added to the sample which is then centrifuged at a high speed. The organelles will form layers according to their specific densities.
Why is it possible to separate organelles using centrifugation?
Because the various cellular components have different sedimentation rates (densities and sizes) ??
