Protocol for preparation of P2 and LP2 fractions

All steps should be carried out at 4°C

Solutions needed:

• 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

Procedure:

1. Wash 20 rat brains in 240 ml ice cold homogenization buffer.
   
   
2. Homogenize brains in 240 ml fresh ice cold homogenization buffer (9 strokes at 900 rpm).
   
   
3. Add 1 : 1000 PMSF solution and centrifuge for 10 min at 2.700 rpm in SS34 or comparable rotor. Discard the pellet.
   
   
4. Centrifuge supernatant (S1) for 15 min at 10.000 rpm in SS34 or comparable rotor.
   
   
5. Resuspend the pellet (P1) in 240 ml ice cold homogenization buffer and centrifuge for 15 min at 11.000 rpm in SS34 rotor.
   The resulting pellet is the P2 fraction and contains enriched synaptosomes.
   
   For the LP2 fraction continue and
   
   
6. Resuspend pellet in 24 ml homogenization buffer.
   
   
7. Split the suspension into to equal portions and transfer into homogenizator. Lyse by adding 180 ml ice cold H₂O to each portion and homogenize 3 strokes at 2000 rpm.
   
   
8. Add 1 ml HEPES buffer and 1 : 1000 PMSF solution and centrifuge for 20 min at 16.500 rpm in SS34 rotor. The pellet (LP1) contains plasma membranes.
   
   
9. Centrifuge the supernatant (LS1) for 2 h at 50.000 rpm 50 Ti. The resulting pellet is the LP2 fraction and contains enriched synaptic vesicles.