Cell Fractionation and Ultracentrifugation
Laura Armstrong
Teacher
Contents
Recall Questions
This topic requires prior knowledge of organelle structure and function. You can test your knowledge on these below.
What is the function of the nucleus in a eukaryotic cell?
The nucleus contains genetic material (DNA) that codes for the sequence of amino acids in a polypeptide. It also contains the nucleolus, which is involved in ribosome synthesis.
What is the role of the mitochondria in a eukaryotic cell?
Mitochondria are the site of aerobic respiration and produce ATP which releases energy.
Describe the role of ribosomes in a cell.
Ribosomes are the site of protein synthesis (where mRNA is translated into a polypeptide chain).
Topic Explainer Videos
Check out this @LauraDoesBiology video that explains cell fractionation and ultracentrifugation or read the full notes below. Once you've gone through the whole note, try out the practice questions!
Overview of Cell Fractionation and Ultracentrifugation
Cell fractionation is the process of breaking up cells and separating their organelles for study under an electron microscope. It involves two key stages: homogenisation and ultracentrifugation.
Homogenisation
- The tissue is broken up using a homogeniser (blender) to release organelles.
- The resulting liquid is called homogenate.
- The homogenate is filtered to remove any large debris and whole cells (that remained intact after homogenisation).
Ultracentrifugation
This process involves spinning the homogenate at different speeds to separate organelles based on their density.
Sample is spun at a low speed to begin with: The heaviest organelles (e.g., nuclei) form a pellet at the bottom, and the rest remain in the supernatant.
Supernatant is transferred and spun at a higher speed: Mitochondria form a pellet.
Repeated at progressively higher speeds: Organelles are separated in order of density:
- Nuclei (largest and densest) - first pellet.
- Mitochondria & chloroplasts (medium density) - second pellet.
- Lysosomes.
- Ribosomes (smallest and least dense) - last pellet.
Why Must the Solution Be Kept Ice Cold, Isotonic, and Buffered?
- Ice Cold: Reduces enzyme activity to prevent the digestion of organelles.
- Isotonic: Prevents organelles from bursting or shrinking due to osmotic effects. Remember an isotonic solution will have the same water potential as the organelles, so will not lead to the movement of water by osmosis.
- Buffered: Maintains a constant pH to prevent protein/enzyme denaturation.
Key Terms
- Homogenisation: Breaking open cells to release organelles.
- Ultracentrifugation: Spinning a sample at progressively higher speeds to separate organelles by density.
- Supernatant: The liquid fraction containing lighter organelles after centrifugation.
- Pellet: The heavier organelles that settle at the bottom after centrifugation.
- Isotonic Solution: A solution with the same water potential as the cell to prevent osmotic damage.
- Buffer Solution: Maintains pH to prevent denaturation of proteins and enzymes.
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Exam Tips
When describing ultracentrifugation, always mention that the supernatant is removed and re-spun at a higher speeds to obtain smaller organelles.
Use correct terminology: ‘organelles’ instead of ‘cell parts’, ‘ultracentrifugation’ instead of ‘spinning’.
When describing why the solution needs to be isotonic or ice-cold, be clear that it is to prevent digestion or bursting of organelles, not cells. The cells have already been broken open during homogenisation!
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Starting with some spinach leaves, describe how you would obtain a sample of undamaged chloroplasts. Use your knowledge of cell fractionation and ultracentrifugation when answering this question. (6 marks)
- Cell homogenisation to break open cells
- Filter to remove large debris / whole cells
- Use isotonic solution to prevent shrinking/ lysis of chloroplasts / organelles
- Keep cold to prevent / reduce digestion by enzymes or use a buffer to prevent protein / enzyme denaturation
- Centrifuge to separate nuclei / heavy organelles
- Re-spin at higher speed to get chloroplasts in pellet / at bottom.
Practice Question
Try to answer the practice question from the TikTok on your own, then watch the video to see how well you did!