7.2.2. High speed centrifugation

1. Centrifuge the supernatant at 75,000 g and 4oC for 3 hours.
2. Discard the supernatant.  
3. Re-suspend the pellet 5 ml extraction buffer.  
This is best achieved by storing the pellet with buffer overnight at 4oC, to loosen the pellet.  
4. Next day vortex the pellet lightly.  
5. Centrifuge at 8,000 g and 4oC for 15 minutes.
6. Retain the supernatant for gradient centrifugation.