I extracted lipids using Folch technique and evaporated the chloroform/methanol mixture and resuspended the lipid in 200-uL of DMSO. I then determined the concentration of triglyceride (mg/dL) using an assay kit. To determine the amount of triglyceride (mg), do I multiply the concentration by the original sample volume (before evaporation) or the resuspended volume. I am slightly confused as I have seen others multiply the original volume.
Dear Bronson,
It is easy, for example you weight at first 100mg liver tissue.
After homogenization, extraction, evaporation and re-suspend in 200ul, you determine the TG using an assay kit. e.g the concentration is 100mg/dl, which is equal 1mg/ml.
So, you have 200ul=0.2ml
concentration is 100mg/dl=1mg/ml
So: 0.2ml*1mg/ml=0.2mg total content of TG
Now, if it is related to 100mg tissue
So 0.2mg/100mg tissue = 2mg/g tissue.
Finally you will have two reports:
1) total TG related to the defined amount of tissue = 0.2mg
2) TG concentration in the tissue is 2mg/g tissue
if you send me the real weight of tissue and final TG concentration using the assay kit, I will send you real calculation.
best
mehdi
Your confusion is understandable. If you determine the triglyceride concentration in the reduced volume (200 µl), you may take the determined "x" mg/dL = "x" mg/100 mL = "x" µg/0.1 mL = "x" µg /100µL = "x"/100 µg/µL, and multiply the "x" by 200, so, you have all triglyceride in the 200 µl, but as µg.
Another question is whether all of the originally extracted triglyceride has been recovered in the 200 µL, but I think you are sure about this,
K.H.
Determine tg in an aliquot
Normalize it to weight. U need to have written down the weight of the tissue which was used for preparing folch extract. Assuming u used all the extract quantitatively, all the triglyceride from the amount of tissue u started with is in 0.2ml of DMSO.
I think you should multiply with the original volume you started with.
Solvebts from Folch extract are usually completely removed by evaporation
You Multiply with the original volume as it represent the all TAG inyour liver samples then you divdie it by the weight of the liver tissue , but the most accurate way is to analyse the protein concentration in your liver samples than the use of the weight .
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