Proteinase K Protection Assay Conditions
Posted 21 April 2009 - 02:59 PM
If anyone has any experience using this technique, a protocol or some advice would be greatly appreciated. If you need more details (concentrations, buffers, how the mitochondria are isolated, the Western blotting info, etc.) please ask.
Posted 23 April 2009 - 04:02 PM
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Posted 24 April 2009 - 07:50 AM
Our lab's technique to isolate mitochondria involves:
Resuspend the cells in isolation buffer (200 mM mannitol, 70 mM sucrose, 10 mM Hepes/pH 7.5, 1 mM EGTA/pH 8) and disrupt with a dounce homogenizer. Pellet the nuclei and unbroken cells (560xg), spin the supernatant at 8000xg to pellet mitochondria and membranes. Wash the pellet 2x with isolation buffer and rususpend the pellet (containing mitochondria) in isolation buffer. We have an optional step to ultracentrifuge the mitochondria to get a cleaner prep but for my purpose I did not think that was important. The main was the Qiagen kit is different is that it has a cell lysis step then further lysis using a syringe and then a lower speed pelleting of the mitochondria (6000xg).