isolation of celmembrane of tissue cells - (Apr/26/2007 )

Hey,
For an application I have to lysis my tissue culture cells (most of the time HEK cells) an isolate the membrane. Can I do this with ultracentrifugation? Which buffer do I have to use? And I can not use detergent.
Thanks in advance!
regards

Yes, you can use ultracentrifuge to collect cell membrane after homogenization. It depends on which membrane are you talking about that would determine the centrifuge speed or how to prepare and collect the sections from sucrose gradient. I don't use salt in my buffer either since you could lose membrane-associated proteins that way. So buffer for me (in case of differential centrifugation to separate plasma membrane, ER, Golgi and other membrane-bound organelle only containing 20mM Hepes, pH7.4, 1mM EDTA, 250mM sucrose). Check for some papers, e.g about GLUT4, David James group. They use that technique alot