Dear colleagues experienced with phospholipid detection technic!
I have a lipid "map" from a cell extract labelled with 32P- isotope resolved by two-dimensional TLC. All phospholipids have been successfully resolved by using non radioactive authentic lipids but I can not find a sample of phosphatidic acid.
"Unfortunatelly" the inhibitors we are going to study may suppress the activity of Phospholipase D so PA concentration have to be measured in the cell.
So I would be very appreciated if someone provide me with the Rf of phosphatidic acid on silica gel TLC in any TLC solvent system.
Hi Dmitry.
It has been many years since I did it, but I used one of the solvent systems in Tysnes et al. Pre-run the silica gel 60 plates in 1% (w/v) potassium oxalate in MeOH/H2O (3:2), spot then pre-activate at 110oC for 20 minutes. Let cool in a dry atmosphere, then develop immediately in CHCl3/acetone/MeOH/H2O/glacial acetic acid (40:15:13:8:12).
He gives the Rf as 0.86, and I measured 0.87+/-0.01 with my samples. There are three other solvent systems described in the link, but I haven't tried them. Good luck!
http://www.sciencedirect.com/science/article/pii/0049384885902683#
Thank you prof. Patterson so much!
I use the system you mentioned for the first direction of 2D TLC elution but have never tried oxalate pre-treatment. Your conditions seems to be perfect because I expected only tiny TLC mobility for PA (
Hi Dmitry,
As you want, you can use our method which is very similar to the suggestion of Prof. Patterson.
I load a power point with the image of yeast PL development.
Good luck,
Thank you prof Rozès !
It's a brilliant resolution for 1D TLC !!!
What kind of TLC plates did you use? And what was the method of sample applying on TLC to get such a perfect thin spots ? It looks like an electrophoresis; I thought what such quality of resolution exist only in educational books and commercial TLC advertisements.
Hi Dmitry,
Thank you a lot for your appreciation but I didn't say all.
I send to you one document where you can find all the details of this method.
The most important thing is (i) sample application to have a good resolution and (ii) a constant temperature (25ºC) and a relative humidity (ca 55%) of the development chamber. You can prove it without the need of ADC2.
Nicolas
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