- Resuspend mitochondria in one-third the packed cell volume mitochondrial lysis buffer (25 mM HEPES KOH pH 7.6, 5 mM MgCl2, 0.5 mM EDTA, 10% glycerol, 1 mM DTT, 1 mM PMSF). Put the suspension into a glass homogenizer and homogenize with 10 strokes using a tight pestle. Add Tween20 and KCl to final concentrations of 0.5% and 0.5 M, respectively.
- Incubate the mixture on ice 5 minutes. The homogenization is repeated for a total of 10 times.
- Spin the final mitochondrial lysate at 100,000 g in an ultracentrifuge using a TY65 Beckman rotor at 4°C, for 60 minutes.
- Carefully collect the clear supernatant, avoiding the fluffy layer over the pellet, to yield the final S-100 fraction.
- Freeze in aliquots, in liquid nitrogen, and store at -80°C.
This is a slightly modified protocol taken from Methods in Enzymology, Vol 264, by Vicente Micol, Patricio Fernandez-Silva, and Giuseppe Attardi.