Free shipping starts now, no minimum, no coupons required!

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 B. Work on ice.
Lysis Buffer B
ReagentConcentration
HEPES (pH 7.4)4 mM
Sucrose320 mM
EDTA (pH 8)5 mM
Phenylmethylsulfonyl fluoride (PMSF)1 mM
Complete EDTA-free protease inhibitor cocktail (Roche)
  1. Homogenize brains with a Polytron homogenizer.
  2. Centrifuge homogenates at 700 x g for 10 minutes at 4°C.
  3. Transfer resulting supernatant to a clean tube.
  4. Centrifuge at 37,000 g for 40 minutes at 4°C.
  5. Discard the supernatant.
  6. Resuspend the pellet (P2) in half the original volume (volume added in step 2) with Extraction Buffer.
Extraction Buffer
ReagentConcentration
Tris (pH 9)50 mM
NaCl150 mM
NP-401%
Sodium deoxycholate0.5%
Phenylmethylsulfonyl fluoride (PMSF)1 mM
Complete EDTA-free protease inhibitor cocktail (Roche)
  1. Incubate at 37°C for 30 minutes.
  2. Centrifuge the solution at 100,000 x g for 60 minutes.
  3. Transfer the resulting supernatant (enriched brain synaptosome) to a clean tube.
  4. Determine protein concentration using the Bradford method and adjust to 4 mg/ml with extraction buffer.
  5. Store samples at -80°C until further use.