Cell Fractionation Flashcards
Density equation
Density = mass/volume
What needs to happen before separating the organelles?
It’s must be broken up and the organelles released and must be in a solution in order to preserve the organelles
What is the perfect conditions for the organelles?
- ice cold
- isotonic
- pH buffered
Why does the solution need to be cold?
deactivates the enzymes found within the organelles so they wont damage the organelles - activity will resume when temp is raised (temporary)
Why does the solution need to be isotonic?
the organelles must be the same water potential as the solution to prevent osmosis as it could cause the organelles shrivel or burst
Why does the solution need to be pH buffered?
the enzymes could denature and not function at the wrong pH causing damage to the organelle
What are the 2 processes in separating organelles?
- homogenisation
- differential centrifugation
What is homogenisation?
cell being broken open to allow the organelles to move out - cell split by mixing solution and tissue in homogeniser (blender)
What happens after homogenisation?
the homogenate is filtered to remove any remaining whole cells or large pieces of cell debris
What is differential centrifugation?
spinning at different speeds
- the homogenate will be spun at low speed to move the densest organelles to the bottom (nuclei)
-the supernatant is poured off and spun at a faster speed which moves the mitochondria to the pellet
-repeated until it moves the least dense to the pellet
What is ultracentrifuagtion?
filtered solution spun at high speeds in a centrifuge to sepereate organelles
What are the most to least dense organelles?
- nuclei
- chloroplasts
- mitochondria
- lysosomes
- endoplasmic reticulum
- ribosomes
