Cell Fractionation Flashcards
What does cell fractionation mean?
Separating different parts and organelles of a cell, so they can be studied in detail.
Processes of cell metabolism (photosynthesis and respiration) have been studied this way/
What is the most common method of fractionating cells?
To use differential centrifugation.
- What is the first step in cell fractionation?
Cut the tissue in ice-cold isotonic buffer.
Why is the cold temperature needed to cut the tissue?
To slow enzyme reactions.
Why is the isotonic buffer needed to cut the tissue?
To stop osmosis, so organelles don’t burst.
Why is the buffer used to cut the tissue?
To stop pH change.
- What is the second step in cell fractionation?
Grind the tissue in a blender to break open cells.
- What is the third step in cell fractionation?
Filter.
Removing insoluble tissue like fat and plant cell walls.
- What is the filtrate now called?
A cell-free extract and its capable of carrying out most of the normal cell reactions.
- What is the fourth step in cell fractionation?
Centrifuge filtrate at low speed.
1000 x g for 10 mins.
This pellets nuclei which can be re-suspended
- What is the fifth step in cell fractionation?
Centrifuge supernatant at medium speed.
10,000 x g for 30 mins.
This pellets mitochondria and chloroplasts which can be re-suspended.
- What is the sixth step in cell fractionation?
Centrifuge supernatant high speed.
100,000 x g for 1 hour.
This pellets ER, golgi and other membrane fragments, which can be re-suspended.
- What is the seventh step in cell fractionation?
Centrifuge supernatant at VERY high speed.
300,000 x g for 3 hours.
This pellets ribosomes which can be re-suspended.
- What is the eighth step in cell fractionation?
Supernatant is now organelle- free cytoplasm.
