How to isolate soluble mitochondrial proteins from mitochondrial suspensions.
Mitochondrial Lysis Buffer
25 mM HEPES KOH, pH 7.6
5 mM MgCl2
0.5 mM EDTA
1 mM DTT
1 mM PMSF
- 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.
- For five minutes, incubate the mixture on ice. The homogenization must be repeated 10 times.
- 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.
- 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.
- Freeze in aliquots, in liquid nitrogen, and store at -80 °C.
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