I need the blood group antigens of the erythrocyte membrane for my research and want to prepare ghosts out of EDTA blood. At the moment I just wash the erythrocytes in 0,9% NaCl, lyse in distilled water and resuspend the pelleted ghosts in isotonic buffer, pH 7,4.
Some of my questions are: Do I have to care about resealing and outside-out orientation? How stable are the blood group antigens? Do I need to add protease-inhibitors, any buffer components, or ions to the lysis buffer? How can I store the ghosts the best way (buffer and storage conditions), is it possible to store them at 4°C or -20°C for several month or to dry ghosts (in a lyophilizer for example)? Is there anything else I should care about, like pH or temperature?
I spent many years preparing red cell IOVs (inside-out vessicles). the membrane skeleton protiens (actin - spectrin - 4.1) tend to be very stable when stored at 4C for 2-3 weeks, after that they begin to show signs of degradation. I seem to remember 5 mM Pi pH 8.0 1 uM PMSF for lysis; then there is a different buffer for making IOVs (0.5 mM EDTA pH 8.0 I believe). There is extensive descriptions of these buffers in the literature - just look up any paper on Band 3, or protein 4.1, ankyrin, glycophorin C. Do not freeze the cells, unless you specifically are trying to reseal some. Once the "skeletal protein matrix" is extracted from the membrane, they are subject to rapid oxidation and proteolysis... (like within a few days you will lose all of protein 4.1) if you do not use reducing agents, store under argon/nitrogen, or purify to a high degree... it gets rather complicated... Good luck!
Hi Ryan,
thank you for your answer, especially for pointing out the influence of EDTA. In the meantime I found the paper by Steck and Kant (1974) which is pretty good in details of ghost preparation and how to seal them outside-out or inside-out.
I also tried to lyophilize my samples (50µl aliquots in 0,9% NaCl, freezing on dry ice) - it works pretty well, is easily reconstituted and it seems as if the blood group antigens survived the procedure. Future will show if they are stable for a longer time...
lyophilization is something I never tried, but I would imagine should work pretty well. It will be interesting to see how many reseal after that process? thanks for the update!!
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