How to study cells - Cell fractionation and ultracentrifugation for AQA A-level Biology

Introduction

This tutorial focuses on cell fractionation and ultracentrifugation, essential techniques for studying cells in AQA A-level Biology. These methods enable the separation of cellular components, allowing for detailed analysis of organelles and their functions. Understanding these processes is crucial for mastering cell biology concepts.

Step 1: Understand Cell Fractionation

Cell fractionation is the process of breaking down cells to isolate their components. This is typically done in several stages:

• Homogenisation: This involves breaking open the cells using physical or chemical methods. Common methods include:

  • Blending: Using a blender to disrupt the cell membranes.
  • Sonication: Using ultrasonic waves to break cells apart.
  • Osmotic Lysis: Placing cells in a hypotonic solution to cause them to burst.

• Purpose: The goal of homogenisation is to release cellular contents while preserving the integrity of organelles as much as possible.

Step 2: Prepare the Homogenate

After homogenisation, you need to prepare the homogenate for centrifugation. This involves:

• Filtering: Remove large debris (unbroken cells and cell walls) using a fine mesh or filter paper.
• Buffer Solution: Add a cold isotonic buffer to maintain pH and prevent enzyme activity, which could degrade organelles.

Step 3: Differential Centrifugation

Differential centrifugation separates cell components based on their size and density through a series of centrifugation steps:

1. Initial Centrifugation:

   • Spin the homogenate at a low speed (e.g., 1000-2000 x g) for about 10 minutes.
   • This will pellet the larger components like nuclei and unbroken cells at the bottom.

2. Collect Supernatant:

   • Carefully pour off the liquid (supernatant) into a new tube without disturbing the pellet.

3. Subsequent Centrifugation:

   • Spin the supernatant at a higher speed (e.g., 10,000 x g) for 10-20 minutes.
   • This will pellet smaller organelles, such as mitochondria and lysosomes.

4. Repeat:

   • Continue this process, increasing the speed for each subsequent centrifugation to isolate smaller components (e.g., ribosomes).

Step 4: Analyze Isolated Components

Once you have isolated the organelles, you can analyze them using various methods:

• Microscopy: Use light or electron microscopes to observe the structure of the organelles.
• Biochemical Assays: Test for specific enzymes or proteins to understand the function of each organelle.
• Genetic Analysis: Investigate the genetic material within the organelles if applicable.

Conclusion

Cell fractionation and ultracentrifugation are vital techniques for studying cells in biology. By following the steps outlined above, you can effectively isolate and analyze cellular components to gain deeper insights into their functions. For further learning, consider exploring practical experiments or additional resources related to cell biology.