Labeled Sphingomyelin Synthesis 合成标记的神经鞘磷脂
Contributor: Suprya JayadevDate: Mar. 11, 1993
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 of reaction by running a few microliters of the reaction mix on TLC (solvent system = chloroform/methanol/ammonium hydroxide @ 60:35:8).
4) Once reaction is complete, dry the mixture as much as possible using nitrogen gas.
--> Should be left with a white precipitate in a small volume of oily, yellow residue.--------------------------------------------------------------------------------
Washing reaction products:
5) Dissolve the residue in 2 ml of chloroform and vortex.
6) Add 1 ml of 5% Na2S2O3·5H2O (sodium thiosulfate) and vortex vigorously.
7) Spin @ 3,000 rpm for 5 min and discard upper, aqueous phase in radioactive waste.
8) Wash organic phase again with sodium thiosulfate.
9) Wash organic phase from step (8) twice with 2 N HCl, discard upper phases in radioactive waste.
10) Wash the acidified organic phase twice with H2O, again discard upper phases in radioactive waste.
11) Dry down washed product and resuspend in 2 ml of chloroform.
--> If a precipitate persists, a little methanol may be added to completely dissolve the material.--------------------------------------------------------------------------------