Hi!
I wanted to perform ultracentrifugation to isolate chylomicrons and VLDL particles from culture media (colorless and low glucose DMEM).
I have some questions regarding the procedure:
- Could I do it just adjunting the density of the medium to 1.12 g/ml and centrifugate for 30min, 4hours and 20 hours?
- How can I know the density of the DMEM medium? just dividing the weight by the volume?
- I read in a protocol that it would be possible to stain the lipids of the lipoproteins by using a solution of Fat red 7b (2 mg/mL Fat Red 7B in 0.1 mol/L NaOH with 5 µLof Triton X-100). since I would like to recover intact chylomicrons and VLDL particles I wondered if this procedure could affect the lipoproteins and their structure.
Thanks a lot!
Hi Patricia, I used to work with lipid particles some time ago involving their cellular endocytosis and degradation.
An old-timer who isolated the lipid particles was very gruff about new techniques - in his words "isolate it by centrifugation and ask questions later".
(sorry, probably not a helpful answer)
- Could I do it just adjunting the density of the medium to 1.12 g/ml and centrifugate for 30min, 4hours and 20 hours?
nope, lipoportien particle payload is not a constant, recommend here isopycnic gradients (palm,2012; Plos Biol)
- How can I know the density of the DMEM medium? just dividing the weight by the volume?
DMEM density? why do you need to know? a rule over the thumb, its aqueous - so its close to water...
Triton X-100!!! - guess i do not comment it further...
hope it helps a bit,
cheers markoB
Thanks a lot!
Ok! no triton!
I thought different "aditives" in DMEM could really have a relevance in the density of it... but easier if I can consider it as water!
Isopynic... you mean create a density gradient? (sorry, I´ve just started to deal with ultracentrifugation... and my knowledge about it is quite limited!)
For isolating only Chylomicrons and VLDL, would be then enough to overlay the media (after adjusting its density) with 1.006 g/ml?
no detergents in general!
you have to decide what is interesting to you, the lipid or protein load. if you aim for lipids than use a salt gradient (palm, 2012, plos biology), in case you aim for proteins than use optiprep medium or equivalent (brankatschk, 2010, JNS). also, most people load their columns bottom up, means your sample is mixed into the most dense fraction (optiprep). however, top down gradients are possible and sometimes even better (your sample is the top fraction). its really important to know first what you want...
good luck - markoB
May I suggest:
Isolation of Serum Chylomicrons prior to Density Gradient Ultracentrifugation of Other Serum Lipoprotein Classes
A. H. M. TERPSTRA
Anal Biochem. 1985 Oct;150(1):221-7.
This may be "an oldie" but works. Do not come near lipoproteins with TritonX-100.
Hope this helps. If not, I can provide more detailed variations...
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