crude plasma membrane

[bik25]

Hi!

I'd need the plasma membrane fraction from rat brain for western blotting. I only want to increase the cocncentration of the membrain proteins in my sample, so it doesnt't have to be too pure. Unfortunately I have no access to an ultracentrifuge. Could anyone give me a good protocol? Thanks!

[postdock]

Hello there, yeah i had this problem too once.

The best way to do this, is to get rid of most of the cytosolic fraction however usually this method (and others even ultra) need to be sure you dont have many unlysed cells.

You can check using a microscope after you lyze. You should see mainly little nuclei and hardly any unlysed cells if you are doing things right. Try using a dounce homogenizer and lysing with 30-40 strokes in lysis buffer. Then check the microscope.

After you are sure there are mainly nuclei and broken cells, then spin as fast as your centrifuge can handle. Do this cold. Usually I could get 15,000 rpms or a bit higher with the cold centrifuge. Spin first for an 1hour or as long as you can without annoying your lab mates.

You should see a fatty area in the middle, a pellet at the bottom and buffer/cytosol everywhere else. Get rid of everything else except the pellet and you can wash again in buffer such as PBS or other, and then spin again to obtain a purer fraction.

Good luck

[bik25]

Thanks for your help