Subcellular Fractionation from Liver or Brain Soft Tissues (FOCUS™ SubCell Kit)
OPTIONAL: Delipidated BSA can be added to 1X SubCell Buffer‐II to the concentration of 2mg/ml for removing free fatty acids prior to processing.
An appropriate amount of protease inhibitor cocktail can be added to the 1X SubCell Buffer‐II just before use.
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.
Perform steps 4-7 on ice.
Transfer the minced tissue to an ice‐cold Dounce tissue homogenizer.
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 sample at 700x g for 5 minutes to pellet the nuclei.
Carefully transfer the supernatant into a new tube.
Centrifuge supernatant at 12,000xg for 10 minutes.
Transfer the supernatant (post mitochondria) to a new tube and resuspend the pellet in 10 volumes of 1X SubCell Buffer II without BSA.
Centrifuge the sample at 700x g for 5 minutes to pellet the nuclei.
Carefully transfer the supernatant into a new tube.
Centrifuge supernatant at 12,000xg for 10 minutes and remove the supernatant.
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 12 can be further fractionated using a variety of gradient and differential centrifugations.
Add 10 volumes of 1X SubCell Buffer‐II and using a loose‐fitting pestle, disaggregate the tissue with 5‐10 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.
