In vitro Reverse Ceramidase Assay

Contributor: Samer El Bawab

Date: February 5, 2001

Reagents:

Buffer A

0.2 M Hepes buffer, pH 7.0

0.4 % Triton X-100

[3H]-palmitic acid

D-e-sphingosine

Preparing Substrate:

Prepare a mix containing 20 uM of sphingosine and 200 uM of [3H]-palmitic acid, in 50 ul buffer A to get ~100,000 cpm.

Vortexe vigorously and sonicate for 1 min.

Assaying enzyme activity:

1) Add enzyme solution (50 ul, from tissues ~50-100 ug of proteins, from cells ~200-400 ug of proteins) to 50 ul of substrate mix and mix gently.

2) Incubate for 1h at 37 C

3) Stop the reactions by adding 2 ml Dole solution (isopropyl alcohol: heptane: NaOH 1 N, 4:1:0.1, by vol), and 10 ug of ceramide as carrier.

adjust the pH first with Tris 1 M (pH 9).

4) Add 1 ml of water and 1 ml of heptane,

vortexe vigorously and centrifuge at 3,000 rpm for 2 min.

5) Collect the upper phase

6) add 2ml of heptane, vortexe vigorously and centrifuge at 3,000 rpm for 2 min.

7) Collect the upper phase, combine with the upper phase from step 5 and count.

NB: this assay should be used only when labeled fatty acid is used.