Preparation of Crude Synaptosomal Fractions from Mouse Brains and Spinal Cords

Materials and Reagents

1. Pipette tips
   10 µl pipette tips (SARSTEDT, catalog number: 70.1130 )
   200 µl pipette tips (SARSTEDT, catalog number: 70.760.002 )
   1,000 µl pipette tips (SARSTEDT, catalog number: 70.762 )
   
2. Reaction tubes
   1.5 ml tubes (SARSTEDT, catalog number: 72.706 )
   2 ml tubes (SARSTEDT, catalog number: 72.691 )
   
3. Mice (Protocol has been tested with male and female C57Bl/6 mice of 8-52 weeks)
   
4. Sucrose (≥ 99.5%) (Sigma-Aldrich, catalog number: S0389 )
   
5. HEPES (≥ 99.5%) (Sigma-Aldrich, catalog number: H3375 )
   
6. Protease inhibitor cocktail (cOmplete^TM Mini EDTA-free EasyPack) (Roche Diagnostics, catalog number: 04693159001 )
   
7. Phosphatase inhibitor cocktail 3 (Sigma-Aldrich, catalog number: P0044 )
   
8. Sodium chloride (NaCl) (≥ 99.5%) (Carl Roth, catalog number: 3957 )
   
9. Potassium chloride (KCl) (≥ 99%) (Carl Roth, catalog number: P017 )
   
10. Di-Sodium hydrogen phosphate (Na₂HPO₄) (≥ 98%) (Carl Roth, catalog number: P030 )
    
11. Potassium di-hydrogen phosphate (KH₂PO₄) (≥ 99%) (Carl Roth, catalog number: 3904 )
    
12. Lysis buffer (pH 7.5) (see Recipes)
    
13. Phosphate-buffered saline (PBS) (see Recipes)
    

Equipment

1. Pipettes (Research 0.5-10 µl, 10-100 µl, 100-1,000 µl) (Eppendorf)
   
2. Heraeus Biofuge Stratos (Rotor) (Heraeus Holding, catalog number: 3332 )
   
3. Ultrasound sonicator (Emerson Electric, BRANSON, model: 150 )
   
4. Stirring device (Ingenieurbüro CAT M. Zipperer, model: R 50D )
   
5. Glass homogenizer (2 ml) (Carl Roth, catalog number: TT57.1 )
   
6. Teflon pestle (Carl Roth, catalog number: TT63.1 )
   

Procedure