In vitro Ceramidase Assay

Contributor: Samer El Bawab

Date: February 5, 2001

Reagents:

Buffer A

0.2 M Glycine buffer, pH 9.5

1 % Triton X-100

Buffer B

0.2 M Hepes buffer, pH 7.0

1 % Triton X-100

Buffer C

0.2 M Acetate buffer, pH 5.0

1 % Triton X-100

[3H]-C16-Ceramide: 1 mM at ~ 100,000 cpm/10 ul

Preparing Substrate:

Resuspend 10 ul of dried [3H]-C16-Ceramide in 100 ul appropriate buffer to get 10 nmoles, 100,000 cpm.

Use Buffer A: for Alkaline ceramidase

Use Buffer B: for Neutral ceramidase

Use Buffer C: for Acide ceramidase

Vortexe vigorously and sonicate for 1 min (make sure all ceramide is fully solubilized).

Assaying enzyme activity:

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

For neutral and acid ceramidase activities 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) Discard the upper phase

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

7) Discard the upper phase and repeat step 6

8) Add 1 ml of sulfuric acid 1 N, 2 ml of heptane, vortexe vigorously and centrifuge at 3,000 rpm for 2 min.

9) Collect the upper phase and count.

NB: this assay should be used only with ceramides labeled on the fatty acid part.