I am following this protocol to produce liposomes. I reconstitute the dry lipids (polar lipids from E.coli) in a buffer with 10% detergent (OG) at a concentration of 4 mg/ml and this is diluted with the same volume of buffer containing my protein or nothing for the liposomes control, now lipid concentration is 2mg/ml and detergent concentration 5%. At this point the mixture is transparent. After 10 minutes, I add the Bio-beads (1g per 1 ml) and leave them stirring at room temperature. After about 30 minutes, the sample becomes cloudy (signal of the generation of proteoliposomes/liposomes (?)) but after a while and before removing the bio-beads, the sample becomes transparent again.
-That could be happening? Does it mean no liposomes have formed?ç
Other times I have done the same with the same protocol and the cloudiness did not disappear.
*I wash the bio-beads (Bio-Beads SM-2 Resin-BioRad) with methanol (2x), water (3x) and buffer (3x). And I add the dry bio-beads to the mixture of lipid-protein-detergent.
Thanks.
Hi Lucia,
Potentially you are removing the detergent to quickly. The initial cloudiness you observe could be related to precipitation and not liposome formation.
The tendency to form precipitate instead of liposmers may dependent on the lipids and proteins used.
To solve the problem what could I do? Should I add the bio-beads little by little?
Thank you very much for your reply Sascha Rexroth .
I do not know for sure, if this will solve your problem, but I used to add Biobeads in portion for 2D crystallization.
An other option would be to start with microdialysis (https://hamptonresearch.com/uploads/cg_pdf/CG101_Microdialysis_Crystallization.pdf) and use Bio-beads in a second step to remove the detergent completely. However, this is much slower, than the method you described and might not be helpful in your case.
- Badges
- Science topic
- Similar topics
- Mathematical Sciences
- Graphs