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Brain Synaptosomal Preparation

This brain synaptosomal preparation is commonly called the “P2” protocol. This protocol enriches the synaptosomal fraction of the brain and generally enables easy detection of synapse-enriched proteins.

  1. Remove brains and flash-freeze in liquid nitrogen. Keep brains at -80°C until further use.
  2. Resuspend the frozen brains in 5 volumes of ice-cold lysis buffer. Work on ice.
  3. Homogenize brains with a Polytron homogenizer.
  4. Centrifuge homogenates at 700 g for 10 minutes at 4°C.
  5. Transfer resulting supernatant to a clean tube.
  6. Centrifuge at 37,000 g for 40 minutes at 4°C.
  7. Discard the supernatant.
  8. Resuspend the pellet (P2) in half the original volume (volume added in step 2) with extraction buffer.
  9. Incubate at 37°C for 30 minutes.
  10. Centrifuge the solution at 100,000 x g for 60 minutes.
  11. Transfer the resulting supernatant (enriched brain synaptosome) to a clean tube.
  12. Determine protein concentration using the Bradford method and adjust to 3 mg/ml with extraction buffer.
  13. Store samples at -80°C until further use.

Buffers & Solutions

Lysis buffer:
4 mM HEPES pH 7.4
5 mM EDTA pH 8
320 mM sucrose
1 mM PMSF
Protease inhibitor cocktail (Complete, EDTA-free, Roche)

 

 

Extraction buffer:
50 mM Tris pH 9
150 mM NaCl
1% NP-40
0.5% sodium deoxycholate
0.1% SDS
5 mM EDTA
1 mM PMSF
Protease inhibitor cocktail (Complete, EDTA-free, Roche)