protein isolation

[alex33]

Hello,

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.

[relaxin]

Cytochrome P450s are a big family of proteins. In general, they are membrane-bound proteins. But soluble forms have also been reported. Please read the following article for details.

http://cfgbc.mf.uni-lj.si/publications/ ... CYP450.pdf

[toxlab]

I am guessing you are using liver tissue or hepatocytes?
Typically these are associated with the endoplasmic reticulum...during standard isolations (similar to what you describe) the ER is dissociated into "microsomes", small membrane pieces closed in on themselves.
Usually, I see a much higher speed spin at the end than what you describe, but yes, they should be in the last pellet.
Are you running any marker proteins on these fractions to ensure you method is working best?
Might want to look around the literature to compare other techniques.