Cyp450 Protein Western Blot

[nawi]

This should be quick for someone who has experience. So, someone in my lab tells me I should try to isolate CYP450 from rat brain vessels, without using an extraction buffer (only homogenise tissue to brake the cells, because he says that buffer could interfere with Bradford reagent), and cetrifugation at 13,000 rpm. I think it is impossible because I need 100,000 rpm centrifugation to get soluble proteins in supernatant (free from ER).
Is it possible? Why would I even do this if it's no point in doing that. In every scientific paper they say the same, I need an ultracentrifuge-which I don't have.

Thank you..
(anyone has a spare ultracentrifuge?)

[admin]

Hi Nawi,

the proteins should still be there regardless of the rpm or equipment used. The only difference will be purity of them, and the ability to conduct further analysis on the samples will be affected depending on the purification method used.

What will you be doing after the CYP450 Isolation?

I agree to get soluble protein free from ER you will need better separation techniques, especially if you are looking to quantify, conduct 2-D Gel analysis or similar.

Can you not use a shared ultra centrifuge from your research building?

[nawi]

Hello,

yes i also think i can isolate the proteins, because I found some scientific paper where they have been using 410 and 1230 g, but it depends of the tissue I am isolating them from. Since I am also using vessels (brain vessels, and aorta) I have high hopes. They used 100 000 g only for the whole muscle sample. and I don't need muscles. They are also using homogenisation buffer that consists of sucrose, EDTA and protease inhibitors, to brake the cells (sucrose is used instead of detergent so it don't interferes with Bradfrod).
Purification method, for example?
After the isolation and determining the protein concentration, I should separate proteins from the vessel homogenates by electrophoresys on a 10% SDS-PAGE gel and transfer them on a nitrocellulose membrane. I have primary and secondary antibodies (for all 4 isoformes of the enzyme CYP450-4A). My main concern was that I won't be able to detect the protein if they stay in the ER, so the antibodies won't bind with them.
No, we don't have an ultracentrifuge in this city where I live, but they said if needed I can go in other city to do it.

[nawi]

p.s. can I use glucose instead sucrose to break cells?