plasma membrane fraction

[sylvie]

Hi,

Please I need your help!
If I centrifuge samples homogenated in tris buffer + sucrose, first at 5000g (5 minutes) and then the supernatant harvested at 30000g (1H30), which cell fractions can I expect to collect in the final pellet?
is this protocol sufficient to harvest plasma membrane? I know ultracentrifugation is recommended for this application but I don't have access to ultracentrifuge.

Thank you very much

sylvie

[admin]

Hello Sylvie,

you can isolate plasma membrane using this method however its quite crude (depending on where you want to publish and also if contamination with other parts of the cell with affect your results/interpretation).

Also, if you are going to use these for other investigations, there may be contamination issues (with other organelles, ER membranes etc) that may affect your results.

I think it pays to get access to (i am sure you can find one in your facility or adjacent ones - and if not you can always send the samples out to another place) an ultracentrifuge as once you do it a few times, you can keep the samples in the -80C, do westerns on them and if need be do other studies on it later.

[sylvie]

Hello,

Thank you very much.
I'm interested by plasma membrane fraction because the protein that I'm studying, is active in oligomeric form. And I would like to show that active form is localized at the plasma membrane whereas the non active (monomeric) form is cytoplasmic.

sylvie