Palmitoyl Sphingomyelin-Palmit

Purification of Demethylated Sphingomyelin

________________________________________________________________________ 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.

Labeled Sphingomyelin Synthesis

Synthesis reaction: 1) Mix purified DMSM with cyclohexylamine and 14CH3I at a ratio of 1/1.1/1.3 in 5 ml of methanol. 2) Allow reaction to proceed at room temperature for atleast 18 hours in the dark. 3) Check completion

Sphingomyelin Mass Measurement

  Protocol: Bligh & Dyer extraction 1) Pellet approximately 1 X 107 cells. 2) Resuspend pellet in 3 ml CHCl3: CH3OH (1:2) and vortex hard. 3) Add 0.8 ml H2O, vortex hard and allow solution to sit at room temperature for atleast 30