Materials and Reagents

1. Fresh rat liver tissue (30 mg)
   
2. HEPES (Sigma-Aldrich, catalog number: H4034 )
   
3. KCl (Baker, catalog number: 0 208 )
   
4. Triton X-100 (ACROS, catalog number: 215680010 )
   
5. EDTA (Baker, catalog number: 1073 )
   
6. Sodium orthovanadate (Sigma-Aldrich, catalog number: S6508 )
   
7. β-Glycerophosphate (Sigma-Aldrich, catalog number: G9422 )
   
8. NaF (Sigma-Aldrich, catalog number: S7920 )
   
9. Protease inhibitor cocktail (complete Mini EDTA-free) (Roche Diagnostics; or equivalent) (Roche Diagnostics, catalog number: 0 5056489001 )
   
10. Protein assay kit (Bio-Rad Laboratories, catalog number: 500-0006 )
    
11. MgCl₂ (Sigma-Aldrich, catalog number: 16104 )
    
12. Sucrose (Sigma-Aldrich, catalog number: 16104; extra pure grade) (see Recipes)
    
13. Phosphatase inhibitor cocktail (Roche Diagnostics, catalog number: 04906837001 ) (see Recipes)
    

Equipment

1. Ultraturrax homogeniser (T 25 digital ULTRA-TURRAX^® IKA^® Werke Staufen/Germany)
   
2. Centrifuge (Ultracentrifuge, Beckman Coulter, model: Optima XE-100; ultra clear centrifuge tubes, catalog number: 344058 )
   
3. SW28 (or equivalent) swing-out rotor [swinging bucket rotor, catalog number: 2352 ; Beckman SW 28 (Beckman Coulter, catalog numbers: 342207 , 342204 ), with titanium buckets 6 x 38.5 ml, 25 x 89 mm, 28,000 rpm, 141,000 x g]
   
4. Bausch and Lomb refractometer (Lab Logistics Group GmbH)
   

Procedure