Phosphoinositide Thin layer chromatography question
(by legatek from hotmail.com)
Fri Mar 9 02:37:27 EST 2007
Laura Saavedra wrote:
> 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!
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.
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