apoptosis by PCR? - (Jun/24/2010 )
Curtis on Jul 3 2010, 12:03 AM said:
Subcellular Fractionation
Subcellular fractionation was performed as previously described
<40> , but with minor modifications. Infected cells were
collected at different time points and resuspended in mitochondrial
buffer (70 m M Tris-HCl, 0.25 M sucrose and 1 m M EDTA,
pH 7.4). An equal volume of ice-cold digitonin lysis buffer (2 mg/
ml, 19.8 m M EDTA, 0.25 M D -mannitol and 19.8 m M MOPS, pH
7.4) was added for 90 s. Samples were then centrifuged twice at
300 g for 5 min to pellet the nuclei. The supernatant was further
centrifuged at 17,000 g for 20 min to separate mitochondria from
the cytosol.
then add RIPA to your nuclei and mitochondria fractions
Subcellular fractionation was performed as previously described
<40> , but with minor modifications. Infected cells were
collected at different time points and resuspended in mitochondrial
buffer (70 m M Tris-HCl, 0.25 M sucrose and 1 m M EDTA,
pH 7.4). An equal volume of ice-cold digitonin lysis buffer (2 mg/
ml, 19.8 m M EDTA, 0.25 M D -mannitol and 19.8 m M MOPS, pH
7.4) was added for 90 s. Samples were then centrifuged twice at
300 g for 5 min to pellet the nuclei. The supernatant was further
centrifuged at 17,000 g for 20 min to separate mitochondria from
the cytosol.
then add RIPA to your nuclei and mitochondria fractions
Thanks! That was quick. You didn't use any kind of protease inhibitor? I usually add the Mini Complete inhibitor cocktail from Roche. It probably won't disturb the protocol if I add it?
-snoopyx-
I added Roche PI Tablets to digitonin buffer only.
-Curtis-
Curtis on Jul 5 2010, 02:29 AM said:
I added Roche PI Tablets to digitonin buffer only.
Thx a lot Curtis for your help.
-snoopyx-