Hello experts,
I want to build a calibration curve by purchasing the lipid mixture containing several group of lipids with different concentration (For example: PC, PE, CE, SM with concentration 100ug/ml, 20ug/ml, 5ug/ml, 40ug/ml). My concern is- Do I need to use individual lipid to make a calibration curve or use mixture of lipid at different dilution to make calibration curve for different lipid in the mixture?
Also does anyone can suggest me if I can use any matrix for my internal standard to make the matrix closer to plasma or serum? I want to make calibration curve by extracting lipid from the matrix rather than just the organic solvent mixture.
Please let me know if my question is not clear.
Thank you.
Sangita
If the PC, PE CE and SM are mixtures, the mixture would have to be thoroughly characterized (%14:0-18:1, %16:0-18:1, %18:0-18:1, %14:0-18:2, %16:0-18:2, %18:0-18:2, and etc.) to be of any use as an external standard. It would be better to use a pure lipid like DPPC and quantitate all the other PC's against this pure standard; then use DPPE for PE's and etc. You can always do relative response experiments to show the accuracy of this method, if pure standards for the other lipids with different side chains are available.
As for matrix we simulated plasma by using phosphate buffered saline with fatty acid free BSA (or HSA) at 20mg/ml.
Thank you Levison and Guo for your suggestions.
Note: @Levison- I have lipid mixtures with their know concentration. I want to use this lipid https://avantilipids.com/product/330707/ for external standard and want to use the same lipid mixture for my internal standard. So instead of choosing individual lipid, I want to use the above mixture for external and internal use.
Protein quantification:
I have seen people quantifying protein content in plasma while extracting the lipids. How important is protein quantification? What method(s) is suitable for protein quantification from small volume of plasma (For example 100 ul)?
Thank you again for your nice suggestions.
Sangita
1. You choose individual parameters on one concentration pure standarts (include LODs).
2. After this you can use mixture to build calibration curve.
The individual parameters depend on the method used.
In my opinion you need different proteins (or mixtures) for external and internal use.