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 .
