Quantitative determination of ceramide using E. coli diacylglycerol kinase

 Harvest the cells (~1 × 10⁶ cells) by centrifugation.

 Add 500 μL of MeOH to the sample, and sonicate the cells to lyse them.

 Add 500 μL of CHCl₃ and 450 μL of distilled water (DW).

 Vortex for 15 sec.

 Centrifuge at 2,000 × g for 5 min.

 Remove the organic phase and dry using a Speed Vac concentrator.

 Re-suspend the dried sample through brief sonication in 20 μL of 10 mM imidazole, pH 6.6, containing 7.5% of n-octylglucoside, 5 mM cardiolipin, and 0.2 mM diethylenetriaminepentaacetic acid (DETAPAC).

 Transfer the sample (20 μL of solubilized lipid or standard Ceramide) to an Eppendorf tube.

 Mix with 60 μL of 20 mM Mops buffer, pH 7.0, containing 50 mM NaCl, 1 mM dithiothreitol, 3 mM CaCl₂ and 10 μL of DGK (5 mU).

 Initiate the reaction by adding 10 μL of [γ-³²P]ATP (5 μCi; 10 mM ATP in 100 mM MgCl₂).

 Incubate at 30°C for 30 min.

 Add 0.6 mL of CHCl₃/MeOH (1:1, v/v).

 Vortex for a few seconds and add 250 μL of 1 M KCl in 20 mM Mops, pH 7.0.

 Centrifuge at 10,000 × g for 5 min.

 Transfer the organic phase to an Eppendorf tube and dry under a stream of N₂ gas.

 Dissolve the sample in an aliquot of chloroform/methanol (1/1, v/v) and spot on a TLC plate.

 Develop the TLC plate with chloroform/acetone/methanol/acetic acid/water (10/4/3/2/1, v/v/v/v/v).

 Dry the TLC plate and cover with a wrap.

 Expose the TLC plate to an imaging plate.

 Determine the radioactivity of the bands corresponding to [³²P]Cer-1-P with an imaging analyzer such as the FLA5000.