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 .
