When isolating LDL from human blood samples we use density gradient ultracentrifugation according to the method of Proudfout et al. (blood drawn in EDTA coated tubes, spin off 10 min. at 3000rpm, adjust plasma density to 1.26 g/mL with KBr, and overlay this plasma with different KBr densities (1,063, 1.020, 1.006g/ ml) and milliQ respectively. After ultracentrifugation we can collect VLDL (white) on top and also the orange LDL and HDL bands in the tubes.
We performed the same experiment for pooled mice blood, but we do not detect the bands after ultracentrifugation (picture in attachment). How is this possible?
Might this be due to haemolysis? (We tried the procedure for plasma, using EDTA coated tubes and for serum letting the blood clot for half an hour, but with both methods we had some haemolyis and no result). Might the lipoproteins be present but not coloured orange as they are in humans? I know that mice have high levels of HDL as compared to humans, but we can not detect HDL nor LDL in the tubes (I do believe I did find some VLDL on top).
apoB-containing lipoproteins in mice are rather white (not orange) and you only find very low amount of LDL in mice fed a chow diet. You should try to determine lipid contents in the theoric layers or, better, try to use sequential ultracentrifigation to isolate them (because you can concentrate the layer of each lipoprotein)
In general, hemolysis is not a problem for the separation of lipoproteins by ultracentrifugation. Indeed, color of lipoproteins of mice is less intense than in humans due to diet. I suggest two alternatives: 1 - use sequential ultracentrifugation; 2 - try to separate lipoproteins 30 days of feeding mice with diet containing 1% cholesterol. Lipoprotein of hyperchlesterolemic mice is easier separate and this profile can be used to normolipidemic mice.
Because LDL level is very low in normal mouse plasma, and mouse LDL is faint white, it is very difficult to see the LDL band in a centrifuge tube. We separated mouse LDL using sequential ultracentrifugation. After removing chylomicron and VLDL, samples adjested to d=1.019 with KBr were centrifuged. Although LDL should be floated up to the top of the tube by this centrfugation, we barely saw LDL layer. Anyway we scooped small volume of the sample from the top. We could detect presence of apoB in this fraction by SDS-PAGE and/or western blot.
Read about Differences betwwen Human and mice, Mice lacks Cholesteryl ester Transfer Protein ( CETP). Mice presents More VLDL and low LDL.
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