I am reconstituting purified proteins in lipid vesicles and then doing gel filtration with G-25 resin to obtain vesicles of only a particular size. However, I found some lipids do not elute out of that column. Can anyone tell me which lipid can form tight vesicles as well as elute properly from the G-25 resin column?
Dear Sir. Concerning your issue about the lipid which is most compatible with size exclusion chromatography using G-25 resin . Size exclusion chromatography is the method of choice for separating free from liposome-encapsulated molecules. However, if the column is not presaturated with lipids this type of chromatography causes a significant loss of lipid material. To date, the mechanism of lipid retention is poorly understood. It has been speculated that lipid binds to the column material or the entire liposome is entrapped inside the void. Here we show that intact liposomes and their contents are retained in the exclusion gel. Retention depends on the pore size, the smaller the pores, the higher the retention. Retained liposomes are not tightly fixed to the beads and are slowly released from the gels upon direct or inverted eluent flow, long washing steps or column repacking. Further addition of free liposomes leads to the elution of part of the gel-trapped liposomes, showing that the retention is transitory. Trapping reversibility should be related to a mechanism of partitioning of the liposomes between the stationary phase, water-swelled polymeric gel, and the mobile aqueous phase. Retention of liposomes by size exclusion gels is a dynamic and reversible process, which should be accounted for to control lipid loss and sample contamination during chromatography. I think the following below links may help you in your analysis:
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1142305/pdf/1472-6750-5-11.pdf
https://brf.anu.edu.au/files/gel_filtration_handbook.pdf
Thanks
Avoid phosphatidylethanolamine, because this lipid does not form closed liposomes. Phosphatidylcholine works well.
Dear Sutonuka Bhar
Equilibrating GFC column with 5% Glycerol to 8% Glycerol might work, I dont know the condtions of your lipid samples to be carried in. But Glycerol reduces the column matrix and molecule interatctions. resolution ll be good with glycerol too.
Are you doing this on AKTA?
If so you can try glycerol and also sucrose 0.2 M in your buffer.
also after concentrating it to 5 or 10 mL for injection just check for aggregates or precipitate, that may be adding up complications. you can filter your samples in 0.2 micronfilters.
Sutonuka Bhar ... Please check references below.. hope it will be helpful for you…
Regards…
https://www.springer.com/cda/content/document/cda_downloaddocument/9781607619123-c1.pdf?SGWID=0-0-45-1028143-p174024872
https://www.gelifesciences.co.jp/catalog/pdf/18112419.pdf
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