Subcellular Fractionation Protocol

1. Homogenize tissue. (Different tissues require different homogenization techniques and often optimization is required.)

2. 1,000g X 10 min. (pellets nuclei, heavy mitochondria, plasma membrane sheets)

3. 3,000g X 10 min. (heavy mitochondria, plasma membrane fragments)

4. 6,000g X 10 min. (mitochondria, lysosomes, peroxisomes, intact Golgi)

5. 10,000g X 10 min. (mitochondria, lysosomes, peroxisomes, intact Golgi membranes)

6. 20,000g X 10 min. (lysosomes, peroxisomes, Golgi membranes, large dense vesicles (e.g., rough ER)

7. 100,000g X 10 min. (all vesicles from ER, plasma membrane, Golgi, endosomes, etc.)