I am trying to separate the cell membrane proteins, mitochondrial membrane and cytosolic proteins from rat heart (for western blot). I want to work with both membrane and cytosolic proteins .
I am intending to use Mem-PER Plus Membrane Protein Extraction Kit (pierce).
My first question is should I use proteinase liquor in extraction kit ?
Second, When I use this kit can we also isolate mitocondrial membrane proteins too ?
or Does anyone have any other suggestions?
Not sure if that works for your type of cells but you could try to make membrane enriched fractions via centrifugation of whole protein extract since it is easy and cheap. You basically just work with different speed of centrifugation. You first make a whole protein extract. Then you centrifuge at for example 3000 rpm to seperate big cell debries. You take of the supernatant and spin that at high speed. The pellet you obtain should contain your membranes and membrane bound protein. The supernatant your cytosolic protein. Its a primitive method but it works very well. However, i am not sure if it will work for your specific research questions. Just a suggestion.
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