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.
