Purification of Demethylated Sphingomyelin
1) Pack a Bio-Sil A column as follows:
a) Pack base of column with a small portion of glass wool.
b) Suspend 20 grams of 100-200 mesh BioSil A in 100 ml chloroform.
c) Pour silica/chloroform mixture into column eluting chloroform from base.
- Press out any air bubbles in the glass wool as the gel is initially being poured. While pouring column matrix, stir continuously with a stir bar in order to prevent bubble formation in the column.
d) Wash column through with "200 ml chloroform.
- ALWAYS maintain a fluid level above the packing silica... NEVER let column dry out!!
2) Dry pooled lower phases from the demethylation reaction using the rotovapor system.
--> DO NOT heat!
3) Resuspend dried lipid in a minimal volume of chloroform and carefully layer over packed column.
4) Allow sample to run into column while maintaining a small level of chloroform above the packed silica.
5) Sequentially elute with the following solvents:
a) 100 ml chloroform
b) 200 ml chloroform/methanol (20:1)
c) 200 ml chloroform/methanol (9:1)
d) 200 ml chloroform/methanol (5:1)
e) 200 ml chloroform/methanol (4:1)
f) 800 ml chloroform/methanol (3:1)
g) 200 ml chloroform/methanol (3:1)
6) Dry down fractions (f) & (g) separately and re-suspend in 4 ml of chloroform/methanol (1:1).
7) Spot 2 l of each fraction and a small portion of DMSM & SM standard onto a TLC plate.
8) Run TLC in chloroform/methanol/ammonium hydroxide (60:35:8) and develop spots using iodine vapor then potassium permanganate spray.
--> Fraction (f) should contain the DMSM.
9) Completely dry down the DMSM containing fraction.
10) Determine gram weight of DMSM recovered.
11) Resuspend in 2 ml of methanol and store at -20°C until needed for labeled SM synthesis