Fractionation of Light and Heavy Mitochondria by Gradient Cushion (FOCUS™ SubCell Kit)
Suspend the mitochondrial pellet in 100µl 1X SubCell Buffer‐II.
Make a step gradient by adding 200µl SubCell Buffer‐V to a centrifuge tube and then overlaying with 200µl SubCell Buffer‐IV.
Gently float the mitochondrial suspension on the surface of the step gradient.
Centrifuge the gradient at 20,000x g for 20 minutes.
Observe the two white bands.
Carefully remove each band to a separate tube.
Dilute the mitochondrial suspensions with equal volume of 1X SubCell Buffer‐II.
Centrifuge the tubes at 12,000x g for 15 minutes and discard the supernatant.
Suspend the mitochondrial pellets with 30‐50µl Working Mitochondria Storage Buffer and keep the suspensions on ice before downstream processing.
The suspensions may be stored on ice up to 48 hours.
