Liposome (or proteoliposome) filed is new to me. I do not know this procedure to produce liposome make sense. What I did is prepare DMPC liposome with and without 100mM magnesium. The results were interesing. DMPC in 20mM Tris (7.5), 20mM NaCl, gave kind of transparent solution, still not sure there is liposome or not. DMPC in 20mM Tris (7.5), 20mM NaCl, 100mM MgCl2, gave milky solution. Do you think I have liposome with both procedures. By the way, I prepared liposome with biobead methods. 200ul of 20mg/ml DMPC in 100mM Sodium cholate, 20mM Tris, 20mM NaCl, and (100mM MgCl2) was applied to 120mg of washed biobead SM-2 and incubated 2 hours at 4'C rotator. After recover solution, I did it again with 80mg of washed biobead SM-2 again with the same way.
I want to ask this procedure makes sense for liposome experts.
Dmpc liposomes are 'easily' formed in water (shaking). Likely, you have a small size population (transparent) or big size populations (milky), as a function of ionic strength (buffers). You could check the sizes by means of Dynamic light scattering or electron microscopy. The classic way to prepare liposome is: disperse lipid(-s) in a organic solvent (homogeneus solution), evaporate the solvent (by N2 flow or rotoevaporate) to obtain a lipid film, hydrate the lipid film with water (or buffer,..), shake to obtain a lipid dispersion. If you want more homogeneous populations or small sizes, or single layer liposomes (unilamellar), you need to apply some 'energy' (sonication, temperature changes, extrusion,..). There are a lot of bibliography.
I agree with the answer above, and would prefer not to use cholate
Cholate and magnesium ions; what happens if you mix just these two ? I could imaging that they at these high concentrations might well form a precipitate.
Mercedes, it is really interesting! Neutral lipid, DMPC, give such different size upon preparation of different ionic strength or divalent cation. I thought that milky one is not normal unilamella liposome because it seem aggregate of lipid with nor particular shape. But I can think that it could be really large liposome after seeing your comment.
Ruud, I wonder that I can prepare liposome such high concentration of lipid without help of sodium cholate. My experience is that I could not prepare DMPC or DPPC liposome at higher than 5mM. It was really difficult to make them with 100nm diameter size by extrusion, even I could prepare it using lipid film and hydration method. Secondly, I could not see any strange thing (e.g., precipitation) happen when I mix 100mM sodium cholate and 100mM magnesium chloride. Why do you think it will result a precipitation?
The way to prepare liposomes using lipid film and detergents can be somewhat different. It seems there is something important meaning. I do not think I can find the answer from literature. Would you please recommend literature that describe the comparison of these methods. I was trying to use film, biobead and desalting methods. I could not distinguish the significant differences in terms of sizes determined by DLS, orientation of membrane proteins and permeability.
Thank you for the comments and allowing me to think another interesting topic.
The interesting Ruud comment is likely related to the possible interaction between sodium cholate (negative charged molecule) with divalent cations such as Mg2+, they could precipitate. Although DMPC is a zwiterionic lipid, the salt content in the buffer could modify the surface properties of liposomes and promote the increasing size of liposomes.
If you go to extrude or sonicate, it is helpful (i.e., necessary) to heat the solution to a temperature over the melting temperature of the lipid (23C for DMPC).
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