I am extracting Lipids from RBC Membranes using the Cholorform/Methanol (2:1) method. After phase separation and consequent evaporation of the organic phase, what would be the best way to confirm the presence of lipids in the tubes? Is there any qualitative method that could just confirm the presence of lipids?
Dear Mr Khan,
With the solvent system you have used (Folch et al., 1959) for the RBC you will certainly have lipids in your extract. I believe your question relates to finding out whether you have the ones you´re interested in.
If that's the case then there is no universal method to quantify lipids in organic extracts. There are some methods that you can try to find out.
If you´re interested in estimating free fatty acids (FFA) then you can use GC-FID separation after derivatization of your extract.
In case you want to confirm the presence of phospholipids (PL) you can use estimate them by spectrophotometric methods (Bartlett et al., Anal Biochem (1970) 36, 159, or by HPLC methods in case you have available in your lab.
If you're interested in checking the broad range of lipid classes extracted then you might want to consider to use TLC with primuline staining agents for their detection.
If in fact you're interested in estimating the cholesterol content then possible the commercial enzymatic test might be best.
Hope this helps.
Best regards,
Ana
Take a piece of aluminium foil, bend it into a tiny pan. Weigh it with a very sensitive balance. Pipet a known volume of your soution onto the pan, e.g. 50 microliters. Dry the solvent under a gentle stream of nitrogen. Keep under reduced pressure (e.g. in a lyophilizator) overnight. Weigh it again, sibtract the original weight. Now you have mass of lipids/50 ul of your solution.
Then run a TLC or a few with different solvenr systems. Include known standards such as PC, PE, cholesterol, SM, PS on other lanes. Then you will also ger an idea of the abundance of main species.
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