I am doing PIPK activity assay using PI4P lipid as substrate. I am doing the activity assays, extract the phsophoinositide lipids and analyze the formation of PI(4,5)P2 using TLC system with a solvent composition: CHCl3:CH3OH:NH4OH:H2O (90V/70V/4V/16V).
My question is that sometimes I have very nice sharp bands corresponding to PI(4,5)P2 and other times doing exactly the same procedure I obtained like a "wave" band of the lipid.
I would like to know what could be the problem? Is it any special requierement for prepearing the chamber? Or what could be the problem affecting the running?
I would really appreciate any suggestion!
LLama Gratis a cualquier PC del Mundo.
Llamadas a fijos y móviles desde 1 céntimo por minuto. [Only registered users see links. ]
Laura Saavedra wrote:
The key here is to use fresh solvent, or at least no more than a couple
of days old. I believe the streaks you see to be the result of
decomposition and separation of the solvents. Before you begin, swirl
the solvent in the tank. If it goes even the slightest bit cloudy from
the mixing, throw it away.
When thin-layer chromatography (TLC) for phospholipids is used and Silica Gel G adsorbent containing calcium sulfate as a binder has been used, positions of phospholipids on chromatoplates depend upon the amount of phospholipids applied, especially in the ceplialin group. This “load effect” can be eliminated by using “basic” Silica Gel G plates without calcium sulfate binder. For phospholipids separation we utilized two different plates : a) “neutral” 20 g silica gel slurried with 45 ml distilled water; b) “basic” 20 g silica gel made into a slurry in 45 ml of 0.001 M Na2CO3. In both cases has been utilized, such as developing solvent, Chloroform:Methanol:Glacial acetic arid:water (50:25:8:4 - v/v) mixture, obtaining a very good resolution in the separation of 6 phospholipids mixture (Sigma - Iysolecithin, sphingomyelin, lecithin, phosphatidyl inositol, phosphatidyl serine, phosphatidyl ethanolamine).