Which protocol i can use to isolate protein from mitochondria (COX IV and cyto c?
Dear Sadia,
here is an easy protocol for preparation of Mitochondria-enriched Fractions from Hela cells:
HeLa cells (5 x 10to6/ml) were harvested and resuspended in 500 µl of sucrose/HEPES buffer (0.25 M sucrose with 100mM HEPES, pH 7.4, in the presence of an anti-protease mixture).
Cells were then treated with 0.007% digitonin, and incubated on ice for 5 -20 min. Check cells on microscope: when plasmic membrane of most of the cells appears a little bit fuzzy (less refractive compare to intact cells), proceed to the next step.
Lysate was centrifuged at 10,000 g for 10 min at 4 °C. After two washes in 500µl sucrose/HEPES buffer (and subsequent centrifugation steps), the resulting mitochondria-enriched pellet was lysed in 100mM Tris, pH 7.5, 0.5% Triton X-100 plus protease inhibitors for 20 min on ice. The lysate was then centrifuged at 10,000 g for 10 min at 4 °C, and the supernatant aliquoted and kept at 80 °C.
Hope it helps
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