Cell fractionation Flashcards
In homogenisation and filtration, what is the first step?
Cut the tissue (liver heart leaf etc) in ice cold isotonic buffer
Why does the solution have to be cold? Enzyme
To reduce the activity of enzymes that break down organelles
Why does the solution have to be isotonic? W
To prevent water moving into cells via osmosis
Why does the solution have to have a buffer?
To prevent organelle proteins from being denatured
What is step 2 in homogenisation and filtration?
Grinding the tissues in a blender
What does grinding the tissues in a blender do?
This breaks the plasma membrane, releasing organelles into the solution
What is the third step in homogenisation and filtration?
Filter the substance
What is removed when the solution is filtered?
Any large cell or tissue debris that aren’t broken up
What does the filtrate contain?
A mixture of organelles
What is the 4th step in cell fractation?
Centrifuge filtrate at a low speed- 1000xg for ten minutes, which removes nuclei and chloroplasts if it is a plant cell.
What is the 5th step in cell fractionation? Medium (MC)
Centrifuge supernatant at medium speed ( 10,000xg for 30 minutes) , which removes mitochondria and chloroplasts.
What is the 6th step in cell fractionation? (L,GB)
Centrifuge supernatant at high speed (100,000xg for 1 hour), which removes lysosomes and the golgi body
What is the 7th step in cell fractionation?
Centrifuge supernatant at very high speed (300,000xg for 3 hours), which removes endoplasmic reticulum and ribosomes
What is left at the end of cell fractionation?
Supernatant is now a n organelle free cytoplasm (diluted cytoplasm)
