Cell Fractionation (New AQA AS/A Level)

Introduction

This tutorial guides you through the process of cell fractionation, a technique used to separate different organelles from cells or tissue samples. Understanding this process is important for studies in biology and biochemistry, especially for AQA AS/A Level students. We'll cover the two key stages: homogenisation and differential centrifugation, explaining the methods and their significance.

Step 1: Homogenisation

Homogenisation is the first stage of cell fractionation, where cells are broken down to release their organelles.

• Prepare the Homogenisation Buffer

  • Use an isotonic solution to prevent osmotic damage to organelles.
  • Include a buffer (e.g., phosphate buffer) to maintain pH.

• Homogenise the Tissue

  • Use a blender or a pestle and mortar to physically break down the tissue.
  • Ensure the process is gentle to avoid damaging delicate organelles.

• Strain the Mixture

  • Use a fine mesh or cheesecloth to filter the homogenate, removing any large debris.
  • Collect the liquid portion, which contains the organelles.

Step 2: Differential Centrifugation

Differential centrifugation separates the organelles based on their size and density.

• Initial Centrifugation

  • Place the homogenate in a centrifuge tube.
  • Centrifuge at a low speed (e.g., 1000 x g) for about 10 minutes.
  • This will pellet the heaviest components, primarily nuclei, at the bottom of the tube.

• Collect Supernatant

  • Carefully transfer the liquid above the pellet (supernatant) to a new tube without disturbing the pellet.

• Subsequent Centrifugation

  • Centrifuge the supernatant at a higher speed (e.g., 3000 x g) for another 10 minutes.
  • This step will pellet smaller organelles like mitochondria.

• Repeat as Necessary

  • Continue this process, increasing the centrifugation speed and time to isolate lighter organelles (e.g., at 10000 x g for lysosomes and at 15000 x g for ribosomes).

Practical Tips

• Always keep samples on ice to prevent degradation of organelles.
• Use a centrifuge that can handle the required speeds to achieve effective separation.
• Label all tubes clearly to avoid confusion.

Common Pitfalls

• Over-homogenisation can damage organelles, so ensure to monitor the process.
• Not maintaining appropriate temperature can lead to enzyme activity and degradation.

Conclusion

Cell fractionation is essential for studying cellular components in detail. By mastering the stages of homogenisation and differential centrifugation, you can effectively isolate organelles for further analysis. This technique has wide applications in research and diagnostics, paving the way for advancements in biology and medicine. Consider practicing these techniques in a lab setting to gain hands-on experience.