cell fractionation and ultracentrifugation Flashcards
what is cell fractionation?
the process where cells are broken up and the different organelles they contain are separated out
what sort of solution is the tissue placed in before the process of cell fractionation?
cold, buffered, isotonic (of the same water potential as the tissue)
why is the solution cold?
reduce enzyme activity that might break down organelles
why is the solution buffered?
keep pH constant to prevent proteins in the tissue from denaturing
why is the solution isotonic?
the solution is of the same water potential as the tissue to prevent organelles bursting (lysis) or shrinking as a result of osmotic gain or loss of water
why do we want to isolate intact organelles in cell fractionation?
to study structure and function of organelles
what are the two stages of cell fractionation?
homogenation and ultracentrifugation
explain what happens in homogenation?
cells are broken up by a homogeniser (blender). This releases organelles from the cell.
what is the resultant fluid from homogenation called and what happens to it?
homogenate
it is filtered to remove any complete cells and large pieces of debris
what is ultracentrifugation?
the process by which the fragments in the filtered homogenate are separated in a machine called a CENTRIFUGE
what does a centrifuge do?
spins tubes of homogenate at high speeds in order to create a centrifugal force
describe the process of ultracentrifugation?
-tube of filtrate is placed in the centrifuge and spun at a slow speed
-the densest organelles, the nuclei, are forced to the bottom of the tube where they form a pellet
-the fluid at the top of the tube (supernatant) is removed, leaving behind just the pellet of the nuclei
-the supernatant is transferred to another tube and spun in the centrifuge at a faster speed than before
what is the acronym for the order of organelles in decreasing density?
Naughty
Cheeky
Monkeys
Love
Eating
Red
Raspberries
what is the order for decreasing densities of organelles?
Nucleus
Chloroplasts
Mitochondria
Lysosomes
Endoplasmic
Reticulum
Ribosomes
