Mitochondrial Isolation and Purification from Mouse Spinal Cord

Materials and Reagents

1. Isoflurane (Isosol) (Vedco, catalog number: 50201 )
   
2. Fatty acid (FA) free bovine serum albumin (Sigma-Aldrich, catalog number: 85041C )
   
3. cOmplete^TM ULTRA protease inhibitor tablets, EDTA-free (Roche, catalog number: 0589253001 )
   
4. OptiPrep^TM density gradient medium (Sigma-Aldrich, catalog number: D1556 )
   
5. Bradford reagent (Bio-Rad, catalog number: 500-0001 )
   
6. Mannitol (Sigma-Aldrich, catalog number: M4125 )
   
7. Phosphate buffer saline (PBS) (see Recipes)
   
8. 1 M Tris-HCl (see Recipes)
   
9. 0.5 M EDTA (see Recipes)
   
10. Buffer M (see Recipes)
    
11. Buffer M + 0.45% FA free BSA + protease inhibitors (see Recipes)
    
12. Buffer M + protease inhibitors (see Recipes)
    

Equipment

1. 10 ml disposable plastic syringe fitted (heat sealed) with a 200 μl pipette tip
   Using a Bunsen burner, heat and partially melt the wide end of a 200 μl pipette tip. Before the plastic has time to harden quickly insert the melted end of the 200 μl pipette tip into the luer-lok of a disposable plastic 10 ml syringe. Let the assembly cool and check the tightness of the seal by running 10 ml of water at high pressure through the syringe/tip assembly.  
   
2. Sharp heavy dissection duty scissors
   
3. Euthanasia jar
   
4. Balance and plastic weight boat
   
5. Tweezers
   
6. 1 ml glass homogenizer with loose and tight pestles (Kimble Chase Kontes) (Fisher Scientific, catalog number: 885300-0001 )
   
7. Table top refrigerated centrifuge
   
8. Ultrafuge
   
9. Thinwall, Ultra-Clear^TM 5 ml 13 x 51 mm ultrafuge tubes (Beckman Coulter, catalog number: 344057 )
   
10. SW55Ti swing-out rotor (Beckman Coulter, model: 342194 )
    
11. Pipettes
    
12. 50 ml conical
    

Procedure