Fractionation of Light and Heavy Mitochondria by Gradient Cushion using the FOCUS™ Mitochondria 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 in 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.
