Tsjerk
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TLC on phospholipids
Dear all,
Does any of you have experience on running a TLC with phospholipids?
My problem is that I have an assay which requires that I have the same amount/concentration of lipids in my negative control as in the sample I'm
measuring (laurdan lipid fluidity measurement). I'm trying to show the difference in membrane fluidity depending on a protein which is supposed to
organize the membrane and thereby influencing the fluidity.
One of the ways I could estimate the amount of lipid in my sample is by running a TLC, but when I tried I just got all of my lipids to run with a RF
of 1 (I also want to see the difference between the different lipids). Apparently my lipids of natural origin run differently than synthetic lipids.
I used methanol/chloroform/water in a ratio of 60/30/1 to run the TLC, and the lipids I tried to separate where ordered from Avanti (http://avantilipids.com/index.php?option=com_content&vie...).
I did try to find a suitable solvent combination online, but that gave no result.
I could try a method that involves burning the sample with concentrated sulfuric acid, hydrogen peroxide and hydrochloric acid to determine the
phosphate content, but I would rather avoid that because I have to do this measurement at least the first 10 times I do the experiment.
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phlogiston
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Your research project is very interesting, it seems to have some overlap with the project I worked on as a PhD, at least in terms of the techniques
involved.
It revolved around the reconstitution of various transport proteins in liposomes to measure the transport of metabolites.
I have a good protocol for the TLC, but I am currently on vacation and will have to look it up at home (will be back in 3 weeks) It is too long ago I
actually did this (and I later switched to GC-MS as it was more convenient and more informative, and I then forgot the details of the TLC protocol).
Do you want to run a 2D or 1D TLC? What classes of phospholipids are you interested in separating?
I also used the method you describe for total phospholipid assay, albeit with a different variation for the destruction step. I don't recall exactly,
I believe I mixed the sample with a concentrated solution of magnesium nitrate (or magnesium perchlorate?) and heated to red heat. It is annoying, but
pretty sensitive. I recall a colleague used to mix the samples with with perchloric acid, followed by heating the samples in a nearly boiling hot
waterbath in glass tubes covered with marbles to avoid excessive evaporation.
Regarding the phosphate assay, I am assuming you will use the ammonium molybdate coloring reagent, and I also recall a useful additional step for the
protocol that stabilizes the color complex. I'll see if I can find that too.
-----
"If a rocket goes up, who cares where it comes down, that's not my concern said Wernher von Braun" - Tom Lehrer |
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Tsjerk
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Hello Phlogiston, thanks for the reply. This research is actually for my PhD!
For me a 1D TLC will be sufficient as I only want to use it to estimate the amount of liposomes in my sample. The reason I want to separate is because
I have some known standards of the separate lipids which I could then use to estimate the sample.
I do also know the concentration of the commercial stock solution I use so I could use that as a standard, but in the future I want to extract the
lipids myself from species of which the extracts are not commercially available, so some practice in separation wouldn't hurt.
The lipids I'm looking at are phosphatidylethanolamine, Phosphatidylglycerol, cardiolipin and 17-18% unknown stuff.
We also thought of adding a membrane impermeable fluorescent dye while preparing the liposomes (capturing the dye inside the liposomes), so I could
check the fluorescence after reconstitution to see how much lipid I lose during the process, but that would interfere with all the downstream
processes so I would have to run an extra sample every time which would also be a waste of my protein which is pretty bitchy to purify.
[Edited on 24-7-2015 by Tsjerk]
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S.C. Wack
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Quote: Originally posted by Tsjerk  | | I just got all of my lipids to run with a RF of 1 (I also want to see the difference between the different lipids). Apparently my lipids of natural
origin run differently than synthetic lipids. |
Argentation?
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aga
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Quick google:
"A form of thin layer chromatography in which a stationary phase consisting of silica coated with silver nitrate is used to separate cis- and trans-
fatty acid derivatives"
Where else could you go and have a forum member 'just know' that ?
SM is amazing, as are you SCW.
[Edited on 24-7-2015 by aga]
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S.C. Wack
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Oh so you do drink a lot. I can't think of anything that could partially support that. Perhaps some results that others are unable to reproduce.
The AgNO3 is not what is being sought; the separations are by number of double bonds, but separations are likely, if you really want them. The droids
being searched for have surely been mentioned in the literature specifically for this separation and perhaps even verified and uses ordinary silica
gel (maybe sodium carbonate instead of AgNO3).
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Tsjerk
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Dear S.C. Wack,
thanks for the suggestion, but argentation is a bit more than what I'm looking for. 1D is perfectly fine for me as I only have to know what the amount
of lipid in my sample is. I'm still wondering how it can be that the natural lipids run different from the synthetic though.
@phlogiston: I would really like to see your protocol as I still didn't figure how to solve my problem. Thanks in advance!
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