Hello,
I'm having a problem that I don't know how to solve. I am following a protocol that I did a year ago and now it does not work. 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 for 2 hours at room temperature. After this the sample should be cloudy (signal of the generation of proteoliposomes) but it is still transparent and I don't really know what happens. Not removing the detergent properly?
*I wash the bio-beads (Bio-Beads SM-2 Resin-BioRad) with methanol and then with water. And I add the dry bio-beads to the mixture of lipid-protein-detergent.
Thanks.
So, the same protocol worked a year ago?
Are your detergent stocks and lipid stocks recent? You can always do some detergent quantification in your sample if you think that there is still some, or you can try dialyzing.
Thanks for your answer Jorgaq Pata.
Finally I have solved the problem and I have already managed to obtain a mixture of proteoliposomes that shows turbidity. Using the described protocol but increasing the drying time of the lipids to avoid chloroform residues. And improving the pre-washing of the Bio-beads (2x 10 ml methanol, 3x 10 ml water and 3x 10 ml buffer).
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