Sphingomyelin Mass Measurement
2N methanolic NaOH
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 minutes.
4) Pellet cellular debris by spinning at 3,000 rpm (in the tabletop centrifuge) for 5 minutes.
5) Transfer solvents to a new tube without transferring any of the pelleted debris.
6) Add 1 ml of CHCl3 and 1 ml of H2O to each sample.
--> Upon addition of CHCl3, a biphasic solution should result.
7) Vortex hard and spin samples at 3,000 rpm (in the tabletop centrifuge) for 5 minutes.
8) Remove the upper aqueous/methanolic phase and transfer the lower CHCl3 phase to a new tube.
--> The CHCl3 phase should constitute 2 ml total volume.
9) Dry down solvent under extra dry nitrogen gas.
10) Resuspend lipids in 1 ml of CHCl3, aliquot 800 l of lipid suspension for base hydrolysis and 100 l for phosphate measurement.
9) Bring lipids up to 1 ml volume in CHCl3.
10) Add 100 l of 2N methanolic NaOH to each sample.
--> The final concentration of base is 0.2N.
11) Incubate samples at 37°C for 1 hour.
12) Neutralize base by adding 100 l of 2N HCl to each sample.
13) Add 2 ml CH3OH and 600 l of water to each sample, vortex hard.
14) Allow samples to sit at room temperature for atleast 30 minutes.
15) Add 1 ml CHCl3 to break phases and vortex hard.
16) Complete the extraction by addition of 1 ml of water, vortex hard.
17) Spin samples at 3,000 rpm (in the tabletop centrifuge) for 5 minutes.
18) Aspirate off the upper H2O/methanolic phase and collect the lower, CHCl3 phase.
19) Dry the lipids down under extra dry nitrogen gas, resuspend them in 75l of CHCl3 , aliquot 10 l for phosphate measurements and spot 50l on a TLC plate.
20) Spot standards (1-100 nmoles SM) and develop plates in CHCl3:CH3OH:Acetic acid:H2O (50:30:8:5).
21) Visualize lipids using iodine vapor, mark, and scrape the SM spots into a 13x100 screw-capped tube.
22) Extract the SM from the silica as follows:
a) wash with CHCl3:CH3OH (2:1) 2 times
b) wash with CHCl3:CH3OH (1:2) one time.
23) Quantitate the extracted SM via a standard phosphate assay.