I want to extract total lipids from kidney samples. Most papers refer to the Bligh and Dyer method as used to do it. In addition, most of the studies use the amount of protein in the homogenate to correct the amount of lipids found. However, I don`t know in which step of this method they remove a sample for protein analysis.
Hi Beatriz, the B&D method uses CHCL3 and MeOH to solubilize lipids, but these conditions denature most proteins and make them insoluble precipitates.
Why do you want a way to measure protein when you are using a method to isolate lipids?
Because the majority of studies that my work was based on, correct the amount of lipids found with the protein content... How do you do? Do you just use the tissue weight?
Hi Beatriz, I have solubilized tissues for lipid content or for protein content.
I have never tried to measure both at the same time because you either want to get rid of the proteins or you want to get rid of the lipids.
The methods used are usually incompatible. Isolating the lipids will denature the proteins and solubilizing the proteins uses detergents to wash away the lipids.
I am afraid that protein denaturation by CHCL3 and MeOH will give you a false reading of the lipid content within the denatured proteins. Most of the lipids will probably be trapped within the denatured proteins.
I found this protocol on JOVE website:
Lipid Extraction and Measurement: The lipid extraction protocol was adapted from the Folch method (26) and modified slightly from Trent et al. (10). Briefly, approximately 100mg of tissue was homogenized in 500 μL of ice-cold PBS using stainless steel beads for 30s in a bead beater homogenizer. From each sample, 50 μL were removed for protein analysis, and 1.5 milliliters of 2:1 chloroform: methanol was added to the rest of the homogenate in a glass test tube and vortexed. Samples were then centrifuged for 10 min at 3,000 rpm at 4oC. The lower organic phase was separated with glass Pasteur pipette and blown dry with nitrogen gas. The dried lipid was then dissolved with 500 μL of 2% Triton X-100 in chloroform, further dried, and then dissolved in 100 μl double distilled water. The sample of tissue lysate put aside was used to assay protein content using Bradford reagent (Bio-Rad) following manufactures instructions. Using the tissue lipid extract, assays for triglyceride were performed using previously described assay for plasma lipid. Lipid measurements were normalized to protein content of each sample or milligram of tissue weight.
Beatriz Pereira Did the protocol work? I am trying to extract lipid from arteries and was having the same question. Would PBS be enough to completely homogenize the tissue?
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