We are currently performing this protocol in tissue obtained from mice (Cortex and hippocampus)
following the next steps:
MSH Buffer :
230 mM Manitol
70 mM Sucrose
5 mM HEPES
pH: 7,4
+protease inhibitor cocktail and a phosphatase inhibitor
+ 1mM EDTA (0,2M)
1 hemisphere of hippocampus in 400 ul of MSH buffer
Centrifugate to 600 g x 10 min 4ºC
-Rescue supernatant and centrifugate to 8000 g x 10 min 4ºC
Rescue pellet ( mitochondrial fraction).
The problem arises when we made western blot to the determination of mitochondrial purification and we still have citolosic proteins ( as observed by b-actin) in the mitochondrial fraction.
Maybe try a sucrose gradient to isolate them better, or just add an extra wash step on your pellet (recentrifuge pellet at 8000 xg and resuspend it again)
- Badges
- Science method