Purification of Demethylated Sphingomyelin

1 post / 0 new
cfish
cfish's picture
Purification of Demethylated Sphingomyelin

Purification of Demethylated Sphingomyelin

I. Lower, chloroform phase:

1) Dry on rotovapor system with house vacuum lines. It is not necessary to dry sample completely, but sufficiently to yield a final volume of ~2 mls.

II. Upper, aqueous phase:

1) Dry on rotovapor system with vacuum pump. Dry as completely as possible.
a) Will be left with with an oil that smells very bad (because of the DMSO)

2) Resuspend in ~2 ml chloroform.

III. Interphase:

1) Dry as much as possible on rotovapor system with house vacuum.

2) Resuspend emulsion in benzene and dry down again using rotovapor and house vacuum.

3) Resuspend the resultant white crystalline like powder in ~5 mls of chloroform.

----------------------------------------------------------------------
1) Run all three of the above samples on a TLC with the solvent system: chloroform-methanol-ammonium hydroxide (60:35:8). Run sphingomyelin and demethylated sphingomyelin standards as well.

2) Stain with iodine and permanganate.
--> Permanganate is more sensitive than iodine in picking up spots.
--> DMSO will be predominant in both the upper and lower phases.

3) Column purify the DMSM.

Link: http://www.musc.edu/BCMB/ceramide/protocols/0032.html