Dear all,
I am trying to understand which is the "golden standard" method to separate lipoproteins from the blood (LDL, HDL, IDL, chylomicrons...). I found some methods online, and apparently everyone uses a different one.
I need to resolve lipoproteins as much as possible and be very precise, without altering the lipoproteins that I am separating.
Any suggestions? Which method are you using and why?
Thank you!
Classically, this is done by centrifugation (https://www.jlr.org/content/21/3/284.full.pdf), if large amounts are needed in zonal rotors (https://www.jlr.org/content/12/2/160.full.pdf). A zonal rotor is, in effect, a soup bowl of titanium steel with a sample volume of about 1.5 L. It is loaded at low speed (3000 rpm), then accelerated to full speed for the run and finally unloaded at low speed again. Nifty technique, but few places have the equipment.
If an ultracentrifuge is not available, precipitation may be used (https://www.jlr.org/content/11/6/583.full.pdf)
https://pubs.rsc.org/en/content/articlelanding/2019/lc/c8lc01134f#!divAbstract
Hi,
Use heparin affinity chromatography resin as a capture purposes with gravity column format, then further apply the resulting eluent from affinity chromatography to BioRad Cprime and or aPrime mix mode (hydrophobic CEX-AEX) resins as intermediate cleanup and resolution.
This tandem approach may help...
Thank you all for your answers!
Each one of you gave me different answers, therefore I am asking: which is the most precise, which allows the best resolution and which do not alter my sample and the structure and nature of the lipoproteins present in my sample?
Ismaiel Tekko do you think that the heparin in the column will interfere with and alter my sample? Do you think that an ion exchange resin might determine the separation of proteins from the surface of lipoproteins?
Jasim Al-saadi This seems a great method, have you used it by yourself, and can you give me some recommendations?
Engelbert Buxbaum unfortunately I cannot use a gradient or precipitation (they will alter my sample). Do you think that ultracentrifugation without a gradient can be a possible alternative?
Since during gradient centrifugation you have no binding of lipoproteins the risk for modification is smaller than with any chromatographic technique (other than gel filtration, which isn't really applicable here). All you add is salt, to increase the density of the medium. Theoretically, it should even be possible to use isotonic gradients with nycodenz or similar, if you are worried about high salt washing loosely attached proteins away. Unfortunately, they are quite expensive.
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