Protocol for preparation of brain lysate, P2, LP1 and LP2 fractions
All steps should be carried out at 4°C
- Homogenization buffer: 320 mM sucrose, 4 mM HEPES, pH 7.3
- PMSF solution: 200 mM PMSF in EtOH
- HEPES buffer: IM HEPES-KOH, pH 7.4
- Wash 20 rat brains in 240 ml ice cold homogenization buffer.
- Homogenize brains in 240 ml fresh ice cold homogenization buffer (9 strokes at 900 rpm).
- Add 1 : 1000 PMSF solution and centrifuge for 10 min at 2700 rpm (870g) in SS34 or comparable rotor. Discard the pellet. The supernatant can be used as total brain lysate.
- Centrifuge supernatant (S1) for 15 min at 10000 rpm (11952g) in SS34 or comparable rotor.
- Resuspend the pellet (P1) in 240 ml ice cold homogenization buffer and centrifuge for 15 min at 11000 rpm (14462g) in SS34 rotor.
The resulting pellet is the P2 fraction and contains enriched synaptosomes.
For the LP2 fraction continue and
- Resuspend pellet in 24 ml homogenization buffer.
- Split the suspension into two equal portions and transfer into homogenizator. Lyse by adding 180 ml ice cold H2O to each portion and homogenize 3 strokes at 2000 rpm.
- Add 1 ml HEPES buffer and 1 : 1000 PMSF solution and centrifuge for 20 min at 16500 rpm (32539g) in SS34 rotor. The resulting pellet is the LP1 fraction and contains plasma membranes.
- Centrifuge the supernatant (LS1) for 2 h at 50000 rpm (225634g) 50 Ti rotor. The resulting pellet is the LP2 fraction and contains enriched synaptic vesicles.