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