I want to isolate RBC Ghosts from 2-4ml blood collected in EDTA tubes. I would be glad if someone could direct me towards a protocol regarding this. It is also to be noted that the point of my concern in all this is to extract the membrane lipids from these RBCs. I therefore am looking to acquire only the membrane from which I would then extract the lipids.
Dear Mr Khan,
I have done this in the past and in order to get the RBC ghosts you'll need to do a number of rapid freeze-thaw cycles. Take the volume of RBC and place it in a falcon tube. Put the falcon tube in liquid nitrogen for 2 min, and then in a water bath at 37C. Repeat this freeze-thaw cycle for 5-10 times, centrifuge and you'll have the RBC ghosts in the supernatant. Collect the uper phase and extract lipids by liquid-liquid extraction (LLE) or other extraction methods. Please note that different solvent systems used in LLE will extract different classes of lipids with different efficiencies. There are a number of works focusing on this issue.
Hope this helps.
Best regards,
Ana
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