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 .
