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.