Fractionation of all lipid fractions
When all lipid classes must be investigated, it is convenient to use a procedure modified from the general one in allowing the recovery of non polar as well as the most polar lipids (gangliosides).
The extraction must be complete for all lipid classes. Tissues will be mixed on a vortex in 5 to 10 vol of chloroform/methanol (1/1, v/v) during 30 min. After centrifugation, the pellet is re-extracted with 5 vol of chloroform/methanol (1/2, v/v). After a second centrifugation, the pellet is re-extracted with 5 vol of chloroform/methanol/water (48/35/10, v/v). The three lipid extracts are pooled and dried under nitrogen with warming.
The residue is dissolved in 2.5 ml of chloroform/methanol/water (60/30/4.5, v/v) and applied as previously described to a Sephadex G25 column to purify the extract.
The purified extract is dissolved in 0.5 ml chloroform which is applied to a silica column as previously described for the General procedure.
1- Non polar lipids are eluted with chloroform.
2- Phospholipids and glycosphingolipids (except gangliosides) are eluted with:
– 1.5 ml of chloroform/methanol/acetone/acetic acid/water (52/8/8/18/4, v/v) and
– 8 ml of chloroform/methanol (4/1, v/v)
The two extracts are combined, evaporated and dissolved in 1 ml chloroform for further separation, if needed, on another silica column as described for the General procedure.
3- Gangliosides are eluted successively with:
– 3 ml of chloroform/methanol (2/3, v/v) eluting monosialogangliosides
– 3 ml of chloroform/methanol/water (65/25/4, v/v) eluting disialogangliosides
– 3 ml of chloroform/methanol/water (60/35/8, v/v) eluting polysialogangliosides.
Details can be found in Dreyfus H et al. (Anal Biochem 1997, 249, 67).