🚀 go-pugleaf

Hi everyone!

I have lysed cells with a buffer containing the nonionic detergent NONIDET-P-40 -NP-40-, final 
conc. is 1% (v) in a 100mM tris buffer solution. It leaves the nuclei on the culture-dish, so 
it's easy to measure the DNA content from the same experiment). After that, I`m doing an assay 
with the solubilized cell membranes and cytosol. Then, I`m extracting the phosphoinositides 
with a modified Bligh-and Dyer-method (Biphasic extraction with Methanol/Chloroform and a 
little HCl), the lipids are trapped in the chloroform-rich phase. And the NP40, too, I believe.
The lipid extract is evaporated to dryness in vacuo, then I redissolve the lipds in a few �l of 
CHCL3/MeOH and spot the on a silica gel TLC plate that is developped in 
CHCl3/MeOH/Water/ccNH4OH (120/98/22/4 v/v). 
This results in a diseaster, when NP40 is in the extracts: the starting spot don't get wet 
first, when I immerse the plate into the solvent. It seems, it is repelled by the material 
there. When I was using other extracts that were not processed with NP-40, there was no problem 
with the TLC. 
Do you have a suggestion for easily removing the NP40 (I cannot pass all my samples through a 
IEX column e.g., there are too many and there's too little sample-material)?
I thought of a pre-run of my TLC plates in a solvent that will bring the NP 40 to the front 
whil not affecting my lipids of interest (they have strong negative charges caused by several 
phosphate groups.
Can you help? ANY comments are appreciated!

Thanks a LOT!


If you reply, please mail your post to me, I don't read all newsgroups I was postinting this 
regularely.


-- 
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Wolfgang Schechinger
Institute for Diabetes Reserch 
Koelner Platz 1
80804 Munich
Germany 
Phone +49 (89) 30 79 31 24
Fax   +49 (89) 30 81 733
email u7k0201@sunmail.lrz-muenchen.de

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