Dear All,
I am studying a protein which might possess sterol binding activity. I plan to prepare some liposome and test whether the protein can bind onto it or nor, but have a lot of questions.
First, there are a lot of protocols online for liposome preparation which do make me lost. Could you please share your detailed protocol with me? The simpler the better.
Is it possible to prepare liposome without cholesterol? The protein I am studying is suggested to be cholesterol binding which means it might not be able to bind onto cholesterol free liposome. I hope to compare its binding activities onto different liposomes.
I noticed that, such as L-α-Phosphatidylcholine from sigma, might be from either soybean or egg, is it any difference for it to be used to prepare liposome?
If I labeled my protein with dye and it is able to bind onto liposome, can I check the result under microscope? What I mean is whether the liposome can keep intact on slide during my watch?
Best regards
Zha
It's possible to prepare liposomes without cholesterol. It should be possible with both soy and egg PC. If you don't want to go with a mixture of PCs of different chain length I would suggest to use pure DOPC.
The easiest way to make liposomes is thin film hydration. An organic solution of lipids, generally in chloroform, is dried down in a glass container to make a dry film. For small volumes (< 1 ml) you can use gaseous stream to evaporate solvents, for larger volumes (> 1 ml) it's better to use rotavap. Then you add an aequous solution to the film to form liposomes. If you just vortex the solution you will end up with multilamellar vesicles, with additional sonication you can make them unilamellar and smaller. But liposomes made with this protocol will end up in a size range between 100 and 1000 nm and can have a wide size distribution over that range. If your goal is to do imaging you might be more interested in making giant unilamellar vesicles (GUVs).
A good place to look for liposome formation protocols, handling lipids etc is Avanti's website: https://avantilipids.com/tech-support
If your main goal is imaging, it might also be interesting for you to look at membrane-coated beads instead of liposomes - it's an easy way to obtain lipid bilayer vesicle of a defined size in a size range that is optimal for light microscopy. They are made by mixing liposomes (formed as mentioned above) with silica beads. This is a good paper to look at: Baksh et al., Nature 2004, 427, 139–141.
Yes， I only want to see whether the protein has lipids binding activity or not. I will read the paper you suggested. Thanks a lot.
Dear Hong-Guang,
I don´t have really an answer for you but I attach two papers which might be of interest. Many cationic lipids that can form microvesicles with (negatively charged) peptides or proteins (and DNA, of course). Most of them are commercial (DOTAP etc.). Perhaps this is the way to go for you! Depending on your plans, also check the recent paper by Grandellis et al. in Planta on plant immunity induecd by a cationic lipid!
Good luck, and tell me if it Works! Martin
Many thanks Martin,
I will go through them and find a method which could be used in my lab.
Hongguang
We had good luck with the TNS method described here:
Article The major nectar protein of Brassica rapa is a non-specific ...
It doesn't use liposomes, but showed some high level of specificity.
Thanks Clay.
I already downloaded the manuscript and will read it, especially the TNS method part. Fortunately, the protein I am working on is different from yours. I hope to complete the experiment about it in one month.
Zha
I believe the focus should not solely be on whether we can create liposomes without cholesterol, but rather on whether they will maintain stability.
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