Most protocols for the isolation of mitochondria start with a low speed centrifugation (300-1000 x g) for 3-10 minutes to sediment bigger fragments (tissue chunks, blood cells, intact cells). The mitochondria remain in the supernatant, which is transferred into a new tube. This supernatant is now centrifuged at a higher speed (8.000-14.000 g for 5-10 minutes) to sediment the mitochondria. The supernatant of this centrifugation step contains the cytosolic fraction.

Depending on what you are looking for you may need to centrifuge the cytosolic supernatant again (20,000 g and higher) to sediment Golgi vesicle, ER/SR and other membrane structures.

Thank you sir Gisela Beutner . I have isolated crude mitochondrial fraction. Now to verify it as a pure mitochondria (not contaminated with cytoplasm) i need to do western blot of both that crude mitochondrial pellet and cytoplasmic fraction. Kindly suggest the speed at which i can get the cytoplasm fraction for this experiment. Also suggest the best suitable loading control for both mitochondrial fraction and cytoplasmic fraction ?

Hello Asha

A good loading control for the mitochondrial fraction is VDAC (voltage-dependent anion channel; for example ab15895 from Abcam). This antibody works also well as a mitochondrial marker protein, because it is located solely in the outer mitochondrial membrane. To test the mitochondrial fraction for cytosolic contamination you could use beta-actin (ab6276 or ab8226 from Abcam).

You can get the mitochondrial and cytosolic fraction by centrifugation of the post-nuclear supernatant.

1. Homogenize tissue and centrifuge at 500g for 5-10 minutes at 4C

2. Transfer supernatant into clean tube and centrifuge for 5-10 minutes at 10-12,000 g.

3. The mitochondria are in the resulting sediment and the supernatant contains the cytosol.