Ganglioside extraction

Description

aqueous samples are used for lipid extraction including cell homogenates, homogenized tissue, plasma
to 0.8 volume parts aqueous sample 2 parts of methanol and subsequently 1 part of chloroform are added to give one phase (C:M:W=1:2:0.8)
the mixture is left for an hour at room temperature with periodical vortexing
if gangliosides are needed, the one phasic solution is divided into two parts; one part is partitioned according to the modified Folch method
to the other part 1.2 parts of water is added for phase separation (final composition C:M:W=1:2:2 v/v/v)
after vortexing the sample is centrifuged at 2000 rpm for 20 min
the upper aqueous phase is separated
the lower chloroform phase is reextracted with theoretical upper phase
the combined upper phases are loaded onto a C18 SPE column after adjusting the KCl concentration to 0.1M
gangliosides are eluted with methanol, evaporated and redissolved in 50 uL C-M=1:1