Assay of glycosphingolipid synthases using HPLC and fluorescent substrates

 Mix 50 pmol of C6-NBD-Cer or C6-NBD-GlcCer with 5 mg (6.5 nmol) of lecithin in 100 μL of ethanol and then evaporate the solvent.

 Add 10 μL of water and sonicate to form liposomes.

 For the GlcT assay, 50 μL of reaction mixture should contain 500 μM UDP-Glc, 1 mM EDTA, 10 μL of C6-NBD-Cer liposome, and 20 μL of an appropriate concentration of enzyme. For the GalT assay, 50 μL of mixture should contain 100 μM UDP-Gal, 5 mM MgCl₂, 5 mM MnCl₂, 10 μL of C6-NBD-GlcCer liposome, and 20 μL of an appropriate concentration of enzyme.

 Stop the reaction by adding 200 μL of chloroform/methanol (2:1, v/v). After a few seconds of vortexing, 5 μL of 500 μM KCl was added and then centrifuged.

 Dry the organic phase, dissolve the lipids in 200 μL of isopropyl alcohol/n-hexane/H₂O (55:44:1, v/v/v), and transferr to a glass vial in an auto-sampler.

 An aliquot of sample is automatically loaded onto a normal-phase column and eluted with isopropyl alcohol/n-hexane/H₂O (55:44:1, v/v/v) for the GlcT assay or isopropyl alcohol/n-hexane/H₂O/phosphoric acid (110:84:5.9:0.1, v/v/v/v) for the GalT assay, at a flow rate of 2.0 mL/min.

 Determine fluorescence using a detector set to excitation and emission wavelengths of 470 and 530 nm, respectively. Peaks of fluorescence peaks can be identified by comparing retention times with those of standards.