Cell fractionation

• What is cell fractionation?

  Cell fractionation is the process used to separate different organelles within a cell based on their size and density.
• Why is cell fractionation important?

  It allows the study of individual cellular components.
• What is the purpose of homogenization in cell fractionation?

  To break open cells and release their contents, creating a homogenate for further separation of organelles.
• What are the key characteristics of the buffer solution used in homogenization?

  The buffer solution is isotonic, cold, and buffered to maintain constant pH.
• What does isotonic mean in the context of a buffer solution?

  Isotonic means it prevents osmotic damage by maintaining equal water potential inside and outside organelles.
• Why is it important for the buffer to be cold during homogenization?

  To reduce enzyme activity, preventing digestion of organelles.
• Describe the process of homogenization.

  Cells are placed in a buffer solution and then mechanically broken down using a blender or homogenizer.
• What is the purpose of filtration in cell fractionation?

  To remove large debris such as whole cells and connective tissue from the homogenate.
• What is ultracentrifugation?


  Ultracentrifugation is the process of separating organelles based on their size and density by spinning the homogenate at extremely high speeds.
• Outline the general process of ultracentrifugation.

  Spin the filtered homogenate at low speed, transfer supernatant, spin at medium speed, repeat at higher speeds, and finally spin at very high speeds.
• What happens to the supernatant after each centrifugation step?

  It is transferred to a new tube for subsequent spinning at a higher speed.
• Which organelles are typically found in the first pellet after low-speed centrifugation?

  Nuclei.
• At what centrifugation speed are ribosomes pelleted?

  At very high speed.
• Why is a buffer solution necessary during homogenization?

  It maintains pH, prevents osmotic damage, and protects organelles from degradation.
• What role does filtration play after homogenization?

  It removes large debris, ensuring that only smaller organelles and soluble components remain for ultracentrifugation.
• What is a homogenate?


  A homogenate is the mixture of cell components produced after breaking open cells during homogenization.
• Define an isotonic solution.

  An isotonic solution has the same osmotic concentration as the inside of the cells.
• What does ultracentrifugation achieve in cell fractionation?

  It separates organelles using different speeds in a centrifuge based on their size and density.
• List some applications of cell fractionation.

  • Studying the function of specific organelles.
  • Conducting biochemical analyses of individual cellular components.
  • Extracting organelles for medical research or other scientific purposes.
• Why is cell fractionation important in cell biology research?

  It helps in understanding how different cellular components contribute to the overall function of the cell.
• Why is it important to keep the buffer cold during homogenization?

  To reduce enzyme activity, which prevents the digestion and damage of organelles.
• What equipment is typically used for homogenization?

  A blender or homogenizer.
• How does an isotonic buffer prevent organelle damage?

  By ensuring equal water potential inside and outside organelles, preventing them from shrinking or bursting.
• What happens to the supernatant after each centrifugation step?

  It is transferred to a new tube for subsequent spinning at a higher speed to pellet lighter organelles.
• Which organelles are typically found in the first pellet after low-speed centrifugation?

  Nuclei.
• At what centrifugation speed are ribosomes pelleted?

  Very high speed.
• Why is a buffer solution necessary during homogenization?

  It maintains pH, prevents osmotic damage, and protects organelles from degradation.
• What role does filtration play after homogenization?

  It removes large debris, ensuring that only smaller organelles and soluble components remain for ultracentrifugation.
• What is the definition of resolution in microscopy?

  Resolution is the ability to distinguish two points as separate.
• What happens to the image quality when magnification is increased beyond a certain point?

  The object appears larger but becomes more blurred.
• What is cell fractionation and why is it necessary?

  • Cell fractionation is the process of breaking up cells to isolate organelles.
  • It is necessary to study the structure and function of various organelles.
• What is the first step before beginning cell fractionation?

  The tissue must be placed in a cold, buffered solution of the same water potential as the tissue.
• Why is the solution used in cell fractionation cold?

  To reduce enzyme activity that might break down the organelles.
• Why must the solution have the same water potential as the tissue during cell fractionation?

  To prevent organelles from bursting or shrinking due to osmotic gain or loss of water.
• What is the purpose of buffering the solution in cell fractionation?

  To maintain a stable pH and prevent changes that could alter organelle structure or enzyme function.
• What are the two stages of cell fractionation?

  1. Homogenation

  • Cells are broken up by a homogeniser to release organelles.
  • The resulting fluid is called homogenate and is filtered.

  1. Ultracentrifugation

  • The homogenate is spun in a centrifuge to separate organelles based on weight.
• What is the result of the homogenation process?

  The cells are broken up, releasing organelles into a fluid called homogenate.
• What happens during ultracentrifugation?


  The fragments in the filtered homogenate are separated by spinning at high speeds to create centrifugal force.
• What is the first step in the ultracentrifugation process for animal cells?

  The tube of filtrate is placed in the centrifuge and spun at a slow speed.
• What happens to the heaviest organelles during the first spin in ultracentrifugation?

  The nuclei are forced to the bottom of the tube, forming a sediment or pellet.