I am running a differential centrifugation to try to isolate membrane, nuclear, and cytosolic proteins all at once. have a question about where the cytochrome P450 enzymes would be...
The protocol is as follows:
(1) Place 200mg (0.2g) tissue samples in 13ml round-bottom tubes.
(2) Add 100uL of PI to 10ml of homogenizing buffer
(3) Homogenize in 5X (w/v) 1mL of homogenizing buffer (containing protease inhibitor, 100μl per 10 ml buffer).(1mL *8samples =8ml)
(4) Take 1-1.5 ml of homogenate and put into a 1.5 ml tube.
(5) Centrifuge at 3000 x g (rcf) for 10 min at 4°C.
(6) Add supernatant to an ultracentrifuge tube.
(7) Add more homogenizing buffer (without protease inhibitor) to fill at least ¾ of tube.
(7) Centrifuge supernatant (in ultracentrifuge tubes) at 33,000 x g for 60 min at 4°C.
(8) Resuspend pellet in 300-500μL buffer. This is the membrane protein fraction. Store at -80°C
(9) From centrifuge the resulting supernatant is the cytosolic fraction; take 1.5 ml.
So there is an initial 3000xg spin followed by a 33,000xg spin. What I'm wondering is after the final spin, would the cytochrome P450s be in the membrane pellet or in the cytosol? A colleague of mine and I had a discussion about this and I was thinking pellet because the cyp's are membrane bound.