Purification of Demethylated Sphingomyelin

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Purification of Demethylated Sphingomyelin

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

Link: http://www.musc.edu/BCMB/ceramide/protocols/0039.html