My assay mixture will contain PI and PI4P. I don't have a ladder for phospholipids. I have PI4P and PI. Can I make an easy-to-use and resolve ladder using this mixture? Is there an easy way to visualize the lipids other than using iodine vapor?
It's been many years since I've done it, Mahmoud, but the classis system for separating PI and PIPs on silica gel 60 HPTLC plates was to pre-run them with 1% (w/v) potassium oxalate in H2O/methanol (3:2 v/v), air dry, then activate the plate at 110°C for 20 minutes before spotting. The mobile phase for the separation run was chloroform / acetone / methanol / glacial acetic acid (40:15:13:12:3 v/v). Reliable separation of PI, PIP and PIP2.
I always used iodine for the visualization, so I can't help you there. Good luck!
We used Chloroform: methanol: acetone : acetic acid : water (50:10:20:10:5 v/v) for similar study. However, in your case instead of (acetic acid-water), the glacial acetic acid suggested by Patterson might work better.
And I just realized that I left off the water from the end of the mix above (that's the 3 at the end of the volumes). Sorry about that.
Dear Mahmoud,
There are several reagents you can use to detect phospholipids (PL) in TLC plates.
I'm not particularly fan of iodine vapours and in my experience I've used primuline solution (dilute 5mg into 100mL acetone/H2O w/v) to detect PL under UV lamp including PI lipids. As the PLs you're interested in have a glycosilated moiety, I would also suggest you try orcinol solution (20mg into 10mL H2SO4 70%), spraying your TLC plate in fumehood and take to oven at 100C (10-15min) and you should have pink-violet coloured spots.
If you do not have either of these reagents available in your lab, there are other options you can try that are suitable for glycolipids (naphtyl ethylenediamine, hydroxytetralone are just few examples). Hope this helps,
Ana
Thank you so much for your help!!! I am trying most of these protocols at the moment,
Hello Mahmoud Shobair Ana Reis Tushar Kanti Ray
Can you give me full protocol to detect phospholipids (PL) or extract lipid from bacteria sample ? My bacteria is Acinetobacter baumannii.
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