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.
