Subcellular Fractionation from Skeletal or Heart Muscle Hard Tissues ( FOCUS™ SubCell Kit )
Use a fresh tissue sample ( obtained within one hour of sacrifice ) kept on ice .
Do not freeze .
Weigh approximately 50‐100mg tissue .
On a cooled glass plate , with the aid of a scalpel , mince the tissue into very small pieces .
Suspend the sample with 8 volumes of 1X SubCell Buffer‐II containing 0.25mg / ml trypsin in a 2ml centrifuge tube .
Incubate on ice for 3 minutes and then spin down the tissue for a few seconds in the centrifuge .
Remove the supernatant by aspiration and add 8 volumes of 1X SubCell Buffer‐II containing 0.25mg / ml Trypsin .
Incubate on ice for 20 minutes .
Add BSA Solution to a final concentration of 10mg / ml and mix .
Spin down the tissue at 1,000 x g for 5‐10 seconds in the centrifuge .
Remove the supernatant by aspiration .
Wash the pellet with 8 volumes of 1X SubCell Buffer‐II without Trypsin , and spin down the tissue for a few seconds in the centrifuge .
Remove the supernatant by aspiration and add 8 volumes of the 1X SubCell Buffer‐ II without Trypsin .
Transfer the suspension to an ice‐cold Dounce tissue homogenizer and using a loose‐fitting pestle , disaggregate the tissue with 5‐15 strokes or until the tissue sample is completely homogenized .
Using a tight‐fitting pestle , release the nuclei with 8‐10 strokes .
Do not twist the pestle as nuclei shearing may occur .
Stand on ice for 2 minutes .
Transfer the homogenate to a centrifuge tube and leave large chunks of tissue fragments in the homogenizer to be discarded .
Centrifuge the lysate at 700x g for 5 minutes to pellet the nuclei .
Transfer the supernatant to a new tube .
Centrifuge it at 12,000xg for 10 minutes .
Transfer the supernatant ( post mitochondria ) to a new tube .
The pellet contains mitochondria .
Suspend the mitochondrial pellet in working Mitochondria Storage Buffer ( approximately 50μl for pellet from ~ 100mg tissue ) and keep the suspension on ice before downstream processing .
The suspension may be stored on ice for up to 48 hours .
Enrichment of other cell organelles : The post mitochondria supernatant from step 17 - 18 can be further fractionated using a variety of gradient and differential centrifugations .
