How do I isolate mitochondria from liver tissues?

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Accepted Answer

Perform cervical dislocation to sacrifice the mouse. Immediately remove the liver and place it in an ice-cold beaker with an extraction buffer. 
Rinse the liver with a fresh cold extraction buffer until most blood is removed (5-6 washes). 
Cut the liver to small pieces in the ice-cold beaker using small scissors until it becomes a homogeneous mixture. 
Transfer minced liver to a Dounce homogenizer, adding 3 ml of cold extraction buffer. 
Gently grind the tissue ten times with one pestle and another ten times with a tighter pestle, avoiding bubble formation. 
Transfer homogenate to a centrifuge tube and make up to 30-40 ml with a fresh cold extraction buffer.
Centrifuge at 700 x g for 10 min at 4°C. Pour supernatant to a new ice-cold tube, discard pellet. 
Repeat the centrifugation at 700 x g, pouring supernatant to a new tube. Centrifuge at 10,000 x g for 15 min at 4°C. 
Discard supernatant, resuspend pellet in ice-cold extraction buffer. Centrifuge at 10,000 x g, discard supernatant, and re-suspend the final pellet in a minimal volume (around 0.3 ml) of extraction buffer or specific experimental buffer.
 Following isolation, calculate protein concentration using standard methods.

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