Subcellular Fractionation of Mouse Brain Homogenates

Materials and Reagents

1. Mice
   
2. PBS
   
3. Phosphatase inhibitor (e.g. PhosStop) (F. Hoffmann-La Roche, catalog number: 04906845001 )
   
4. HEPES
   
5. EDTA
   
6. Distilled water
   
7. Glucose
   
8. Protease inhibitors (e.g. Complete) (F. Hoffmann-La Roche, catalog number: 11697498001 )
   
9. Solution A (see Recipes)
   
10. 2.5 M sucrose stock (see Recipes)
    
11. Sucrose solution (0.8 M and 1.6 M) (see Recipes)
    

Equipment

1. A pair of scissor
   
2. A brain scooper
   
3. Falcon tubes (15 ml)
   
4. Centrifuge
   
5. Ultracentrifuge
   
6. Ultracentrifuge rotor (T70.1 Ti and Sw41Ti )
   
7. Ultracentrifuge tubes for T70.1 Ti rotor (Polycarbonate, 16 x 76 mm) (Beckman Coulter, catalog number: 355651 )
   
8. Ultracentrifuge tubes for SW41 Ti rotor (14 x 89 mm) (Seton Open-Top Polyclear centrifuge tubes, catalog number: 7030 )
   
9. Thomas^® Teflon Pestle (A.H Thomas Co., model: 3431-E20 )
   
10. 1 and 10 ml syringes
    
11. Needles (18 and 27 G)
    
12. Gradient Master 107 ip (BioComp, model: 107 ) with marker block and cannula
    
13. Refractometer
    

Procedure