Using the Soluble (S-100) Mitochondrial Fractionation Protocol

How to isolate soluble mitochondrial proteins from mitochondrial suspensions.

Required Materials

Mitochondrial Lysis Buffer

25 mM HEPES KOH, pH 7.6
5 mM MgCl2
0.5 mM EDTA
10% glycerol
1 mM DTT


  1. Mitochondria should be re-suspended in a third of the packed cell volume using a mitochondrial lysis buffer. After depositing the suspension in a glass homogenizer, you can homogenize it by using a tight pestle for 10 strokes. To a final concentration of 0.5% add Tween 20. Also add KCI to a final concentration of 0.5 M.
  2. For five minutes, incubate the mixture on ice. The homogenization must be repeated 10 times.
  3. The final mitochondrial lysate must be spun at 100,000 xg inside of an ultracentrifuge for one hour, using a TY65 Beckman rotor at 4 °C.
  4. Collect the clear supernatant with care, while avoiding the fluffy layer over the pellet, carefully collect the transparent supernatant to yield the final S-100 fraction.
  5. Freeze in aliquots, in liquid nitrogen, and store at -80 °C.

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Last updated: Jan 30, 2020 at 5:40 AM


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