Hi!
As I mention in a previous question I am not a "lipid expert", neither any college in my lab, but I came to a point in the research for my PhD thesis, where I need to extract and work with glycolipids… and I am a bit lost!
I got a protocol to do it through Folch patitioning method. I would need to extract glycolipids from a sample of cow skeletal muscle tissue (no cell culture) I tried to find information about the sample preparation but I just got more confused.
I have doubts regarding the amount of sample I would need to obtain a "decent" amount of glycolipids after the extraction. I read about using no more than 1g of tissue but I don´t know if that would be enough.
Another thing I read is that samples were treated previously with acetone (overnight "incubation"). Would be that needed in case of the kind of sample I have? I thought about homogenizing it by freezing it with liquid N2 and then grind it with a mortar... Again I don´t know if that would be the more appropiate way
Thanks a lot!!!!
Dear Patricia,
You will certainly extract glycolipids (GL) from muscle tissue using the Folch method, as you will extract if you use other solvent systems. Are you simply interested in extracting GL for further or extracting the most of GL from your sample? If the former then Folch is suitable, if the latter then my suggestion is you conduct an experiment using several solvent systems and quantify GL in each extract (for instance spectrophotometrically using orcinol method) to determine which is the most efficient solvent system. I did a similar experiment on LDL to determine the most efficient solvent system (see manuscript attached).
This brings to your next question, what is a "decent" amount of GL for you? Are you thinking grams? mg? Are you planning on using the GL extract to do additional experiments on cells? If that's the case then you're right you'll need a "decent" amount of GL amount to conduct a number of experiments (with replicates). In that case I suggest using the grams of tissue in each extraction procedure and repeat it a couple of times.
I'm not sure about the acetone-step but generally that step is for protein precipitation purposes.
Hope this helps,
Ana
Folch will most likely extract glycolipids, but it would be interesting to be sure by using TLC of the extract: some lipids could potentially remain in the interphase between the 2 solvents after extraction.
As far as the amount is concerned, it strongly depends on the method you are going to follow subsequently for glycolipids analysis. The amount of gangliosides and cerebrosides in muscle must be quite tiny, as far as I can remember from out TLC plates when analysing pork phospholipids.
https://www.researchgate.net/publication/220036718_Improvement_of_solid_phase_extraction_method_for_separation_of_animal_muscle_phospholipid_classes
The amount of sample can be increased, as long as you keep the sample to solvent ratio:
https://www.researchgate.net/publication/222271833_Comparison_of_different_methods_for_total_lipid_quantification_in_meat_and_meat_products
Article Improvement of solid phase extraction method for separation ...
Article Comparison of different methods for total lipid quantificati...
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