Proteins

How we extract proteins from cell organelles:

1. Cell Disruption: First, you need to break open the cells to release their organelles. This can be done using various methods:

   • Mechanical disruption: Homogenization using a Dounce homogenizer, bead beater, or sonication can break open cells.
   • Chemical disruption: Non-ionic detergents like Triton X-100 or NP-40 can selectively disrupt the plasma membrane while leaving organelle membranes intact.

2. Differential Centrifugation: After cell disruption, you perform a series of centrifugation steps to separate organelles based on their size and density:

   • Low-speed centrifugation (around 1,000-2,000 × g) removes unbroken cells, nuclei, and large cellular debris.
   • Higher-speed centrifugation (around 10,000-20,000 × g) separates organelles such as mitochondria, lysosomes, and peroxisomes.
   • Ultracentrifugation (over 100,000 × g) can further isolate organelles like microsomes, endoplasmic reticulum (ER), and Golgi apparatus.

3. Organelle Enrichment: After differential centrifugation, you will have organelle-enriched fractions. However, these fractions may still contain contamination from other organelles. Additional purification steps may be required, such as using density gradient centrifugation or specific organelle isolation kits.

4. Protein Extraction: Once you have isolated the organelles, you can extract proteins from them. This typically involves:

   • Disrupting the organelle membranes using detergents or other membrane-disrupting agents.
   • Releasing the proteins into a buffer solution that stabilizes them and maintains their native structure and activity.
   • Optionally, removing any insoluble material (e.g., membranes, debris) by centrifugation.

5. Protein Quantification and Analysis: After protein extraction, you can quantify the amount of protein using methods such as Bradford assay, BCA assay, or UV spectrophotometry. You can then analyze the extracted proteins using techniques like SDS-PAGE (polyacrylamide gel electrophoresis) or Western blotting to visualize specific proteins or assess their expression levels.

6. Storage: Store the extracted proteins at appropriate temperatures (-20°C or -80°C) to maintain their stability for future experiments.