Glycosphingolipid analysis

1) Incubate cells with 1 µCi/ml of 3H-galactose for 72 hours.
---> If treatment is for an extended period of time: treat in serum free media containing labeled galactose.

2)Wash label out and spin cells down.

3) Extract lipids by washing 3 times with chloroform-methanol (1:1).
---> Use 3 ml each time.

4) Pool the solvents from the extractions and dry down lipids.

5) Resuspend lipids in 2 ml of 0.1 N methanolic sodium hydroxide.

6) Allow base hydrolysis to proceed for 1 hour at 37 °C.
---> Base hydrolysis gets rid of any contaminating phospholipids.

7) Neutralize base by adding 267 µl of 0.75 N HCl (an equimolar concentration of acid to base).

8) Add 1 ml of chloroform to bring the solvent ratio to 3:6:0.8 of chloroform-methanol-water.
---> NOTE: It is best not to extract at this point because gangliosides could potentially partition into the aqueous phase and be lost.

---> This mixture can be stored in the freezer for short periods of time.

9) Prepare a slurry of DEAE-Sephadex in chloroform-methanol-water (30:60:8).
---> This resin has a positive charge and will therefore bind the acidic glycosphingolipids. Thus, when a combination of neutral and acidic lipids are added, the acidic lipids will stick and the neutral lipids will wash through.

10) Pack a Biorad dispo column with 4 ml of packed gel, use chloroform-methanol-water (30:60:8) to pack column.

11) Apply sample to column, wash the sample tube 2 times with 2 ml of chloroform-methanol-water (3:6:0.8) and apply that as well.

12) Elute column as follows:

a) 30 ml of chloroform-methanol-water (30:60:8)
---> This will continue to elute the neutral glycosphingolipids.

b) 30 ml of chloroform-methanol-0.8M NaOH (30:60:8)
---> This should elute the acidic glycosphingolipids.

13) Dry each fraction in the rotovap, resuspend lipid in a minimal volume of the same solvent and transfer to a small screw-capped tube.

14) Dry down samples again and:

a) Neutral glycosphingolipids: Resuspend in a minimal volume of chloroform-methanol (1:2), apply to TLC and develop in chloroform-methanol-water (65:25:4).

b) Acidic glycosphingolipids: Resuspend in 2 ml of methanol-1.6 M sodium acetate (1:1), desalt and apply samples to TLC.