Journal of Lipid Research, Vol 30, 775-779, Copyright © 1989 by Lipid Research, Inc.
High performance thin-layer chromatography and densitometry of synaptic plasma membrane lipids
WG Wood, M Cornwell and LS Williamson
Geriatric Research, Education and Clinical Center, Veterans Administration Medical Center, Minneapolis, MN 55417.
Previously, it has been shown that phospholipids, cholesterol, and
glycolipids could be quantitated using the same high performance thin-
layer chromatography (HPTLC) method. Here we examined that method in terms
of linearity of standards in the nanogram range, recovery of nonacidic and
acidic lipids after Sephadex column chromatography, and quantitation of
lipids in mouse synaptic plasma membranes (SPM) where lipid content is low.
Nonacidic and acidic fractions were separated by Sephadex column
chromatography, applied to plates using contact spotting, chromatographed,
visualized with cupric acetate, and quantitated using in situ densitometry.
Recovery of nonacidic and acidic fractions off the columns was determined
with radiolabeled phospholipids. Standards for each lipid class were linear
in the nanogram range. Quantitation of SPM lipid classes could be made with
as little as 1.5 micrograms of total lipid. Recovery of the nonacidic
fraction after Sephadex column chromatography was approximately 100%
whereas the acidic fraction was approximately 91%. Phospholipids,
cholesterol, and glycolipids could be determined in nanogram amounts using
the same method. This method is an efficient method for examining different
lipid classes and in samples where lipid content is low.
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