I am using Bligh and Dyer method for lipid extraction from cultured cells. Then quantification of Sphingomyelin in the extracted samples using SM assay kit (Fluorometric).
Which solvent is the best to dissolve lipids? I used Choloroform, Butanol and hexane. and all are not miscible with the reaction buffer. DMSO with sonication did not completely dissolve.
At which step we should measure protein to normalize SM content?
Is there another way (other than protein) to normalize the SM content to?
Allmost all the extraction procedures lead to protein precipitation and the solvents are usually not miscible with aqueous buffers. You may extract yours cells with a mixture of chloroform and methanol, then dry your lipid extracts under nitrogen and dissolve again lipids in methanol which is now miscible in aqueous solution. There are also treatments to extract membrane lipids from cell suspensions by using SDS solutions. You should probably test which procedure would extract the higher proportion of your SM. You may normalize your SM content by counting the number of cells in your suspension, by measuring protein content in an aliquot of your cell sample (i.e. at the beginning of the extraction process) or using cell DNA content (fluorometric method) again on an aliquot of your cell suspension.
Cloroform:methanol 2:1 is a very good solvent of SM but you should test if it is miscible with the kit buffer.
Regarding normalization, protein should be measured in the cell homogenates that are prepared before being submited to lipid extraction.
Thanks Begona Ochoa for your concern.
I tried Chloroform: Methanol and other organic solvents.
They are not miscible with the reaction buffer.
Regards
Commercial kits seem to dissolve and prepare the standard sphingomyelin in a detergent solution (2% Triton X-100 in ethanol) then use 10 microlitres per well. I would dissolve my dried Bligh Dyer extracts in this solution too, then use 10 microlitres per well .
Dear Dr Beth Binnington, Thanks for your concern and your valuable answer. But, in my case, I will measure SM which is part of the lipid rafts (Lipid rafts are solubility-resistant to Triton X-100). I do not know If it will work or not. Anyway, I will try your suggestion. Thanks so much.
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