Lecture Notes: Cell Fractionation and Ultracentrifugation

Introduction

Topic: Studying cells and organelles
This lecture covers the second method of studying cells - cell fractionation and ultracentrifugation.
For further learning, refer to previous video on microscopes.

Purpose: Isolate and examine organelles’ structure and function.
Importance: Allows detailed study of organelles, complementing microscopic studies.
Process Involves:
- Preparing cells and organelles in a specific solution.
- The solution must be cold, isotonic, and buffered.

Cold:
- Reduces enzyme activity to prevent organelle damage by enzymes released from lysosomes.
Isotonic:
- Prevents osmosis which could cause organelles to shrivel or burst.
- Important to note: Focus on organelles, not cells, as they are already broken.
Buffered:
- Prevents sudden pH changes that could damage organelles.

Step 1: Homogenization
- Cells are broken open using a blender.
- Use a cold, isotonic, and buffered solution.
- After blending, filter the solution to remove debris.
Step 2: Ultracentrifugation
- Filtrate placed in centrifuge, spinning at high speeds.
- Centrifugal forces separate organelles by density.
- Differential centrifugation applied; repeated spins at increasing speeds.
- Order of Separation:
  - Nuclei (most dense, first pellet)
  - Chloroplasts and Mitochondria (second pellet)
  - Lysosomes and Endoplasmic Reticulum (third pellet)
  - Ribosomes (least dense, final pellet)

Two-Stage Process:
1. Homogenization: Break open cells.
2. Ultracentrifugation: Spin filtrate to separate organelles by density.
Preparation: Use cold, isotonic, buffered solution to avoid damage.
Differential Centrifugation: Organelles separated based on density; denser organelles require slower speeds.

For Practice: Visit Miss Esther.com for questions related to the topic.
Previous Videos: Links available for additional learning on microscopes and other related topics.
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