Subcellular Fractionation from Animal Cells ( FOCUS™ SubCell Kit )
OPTIONAL : Add appropriate protease inhibitor cocktail ( e . g .
G‐Biosciences’ ProteaseArrest , Cat . # 786‐108 ) to SubCell Buffer‐I immediately prior to use .
Use fresh cells only .
Pellet the harvested cells by centrifugation at ~ 800 x g for 1 minute .
Carefully remove and discard the supernatant .
Gently vortex to suspend the cells and incubate on ice for 10 minutes .
Add 500µl of ice cold SubCell Buffer‐I .
Perform this lysis step on ice .
Using a narrow opening ( 20 gauge ) syringe needle , gently pull the suspension up and down 10‐30 times .
Add 250µl 3X SubCell Buffer‐II ( 350µl if Dounce homogenizer is used ) and mix by inverting .
Centrifuge the tube at 700x g for 10 minutes to pellet the nuclei .
Transfer the supernatant to a new tube .
Centrifuge supernatant at 12,000x g for 15 minutes .
Transfer the supernatant ( post mitochondria ) to a new tube for further processing .
The pellet contains mitochondria .
Add 500µl 1X SubCell Buffer‐II to the pellet , and centrifuge again at 12,000 x g for 5 minutes .
Discard the supernatant .
Resuspend the mitochondrial pellet with 50‐100µl Working Mitochondria Storage Buffer and keep the suspension on ice before downstream processing .
Enrichment of other cell organelles : The post mitochondria supernatant from steps 8 - 9 can be further fractionated using a variety of gradient and differential centrifugations .
