I am needing to create liposomes from 3 phospholipids for use with membrane associated cytochromes. Thus far manufacturers instructions (Avanti polar lipids) has not yielded acceptable results. Can anyone recommend a book, review or protocol paper that highlights in detail various methods associated with liposome formation?
It may help if you can give some more details:
1. Are these straight liposomes, proteoliposomes, or liposomes encapsulating something else?
2. Are these small unilamellar vesicles, large unilamellar vesicles, giant unilamellar vesicles,or multilamellar vesicles?
3.What phospholipids are you using?
4. How do you define unacceptable results?
These are lyophilised synthetic lipids: delta-9 Cis and 12:0 phosphocholines and 12:0 phosphoserine. As for the size/type of vesicle, there is no information on that. What information I have is that the cytochrome reductase and cytochrome b5 are placed into a 1 mg/ml lipid concentrate, stood on ice for 5 minutes, 5 mg/ml CHAPS is then added and stood on ice for 5 minutes. Then my enzyme of interest (which the cytochrome reductase system is meant to be supporting via reduction of the enzyme), my enzymes substrate and NADPH is then added. They report 10-20% turnover of my enzymes substrate in 2 minutes. Thus far I have managed 5-10% turnover over 15 minutes - in a cytochrome concentration independent manner. Hence unacceptable results. My problem is that the methods from the original paper are somewhat sparse and refer back to earlier papers which don't actually contain the specific steps used to create the liposomes and protein mixtures.
Jorg, after hydration should the lipids not be always maintained above their Tc? During my sonication I used additions of ice to maintain the water bath at 25 +/- 1 C (Avanti could not provide the Tc of all the lipids and recommended this temperature). We do not have a tip based sonicator.
Tobais, I have checked all proteins for activity using non-liposome assays, and they do work within expected parameters.
A bath sonicator is not always strong enough to produce SUVs. I recommend the extrusion method as it also produces more stable LUVs. The unilamalerity of the vesicles can be checked using NBD headgroup labeled lipid with dithionite. The Tm can be established by differential scanning calorimetry, DPH (1,6-Diphenyl-1,3,5-Hexatriene ) fluorescence anisotropy, or 31P NMR.
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