Proteinase K Protection Assay Conditions - (Apr/21/2009 )

I am looking for a protocol for a proteinase K protection assay to examine which subcompartment a mitochondrial protein resides in. I have been using isolated mitochondria and treating them with proteinase K but for some reason when I look by Western blot I am loosing my inner membrane protein signal. For some reason the proteinase K is only digesting the inner membrane marker protein. I would have assumed that I would be losing the outer membrane protein is anything at all. I thought my purified mitochondria might be broken due to this but I would have expected other mitochondria marker proteins (outer membrane, intermembrane space or matrix) to be lost due to proteinase K digestion. I thought proteinase K should be unable to enter intact mitochondria so I am really confused.

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.

Let us know how the mitochondria are isolated, this way we can tell if there may be a step that's compromising the outer mitochondrial membrane.

-Carlton

I have tried two different protocols to isolate mitochondria: Qiagen's Qproteome Kit (see Qiagen website) or our lab's isolation procedure (see below). I have also tried two different cell types; 143B and Vero.

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).