Isolation of Mitochondria from Soft Tissues ( Liver or Brain ) using the FOCUS™ Mitochondria Kit
OPTIONAL : Delipidated BSA can be added to 1X SubCell Buffer - II to the concentration of 2mg / ml for removing free fatty acids prior processing .
An appropriate amount of protease inhibitor cocktail also 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 this step 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 lysate at 700x g for 5 minutes to pellet the nuclei .
Carefully transfer the supernatant into a new tube .
Centrifuge supernatant at 12,000x g for 10 minutes .
Remove the supernatant and resuspend the pellet in 10 volumes of 1X SubCell Buffer - II without BSA .
( Centrifuge as in steps 9 - 10 . )
Transfer the homogenate to a centrifuge tube and leave large chunks of tissue fragments in the homogenizer to be discarded .
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 .
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 .
( Centrifuge as in steps 9 - 10 . )
Centrifuge the lysate at 700x g for 5 minutes to pellet the nuclei .
Carefully transfer the supernatant into a new tube .
Centrifuge supernatant at 12,000x g for 10 minutes .
Remove the supernatant .
The pellet contains mitochondria .
