Measurement of IPS-PLC activity (Silvia, Yasuo)

1. Collect the cells and remove the medium and wash cells with water.

2. Resuspend cells in 0.25 ml lysis buffer (25 mM Tris-HCl, pH 7.4/ 5 mM EDTA, 20 µg/ml chymostatin, leupeptin, antipain and pepstatin and 1 mM PMSF) and add 0.25 ml glass beads.

3. Homogenate the cells using beads beeter (30 sec, three times).

4. Spin homogenate at 2,500Xg for 10 minutes at 4°C.

8. Collect the supernatant.

9. Centrifuge the supernatant at 2,500Xg for 10 minutes at 4°C.

10. Collect the supernatant.

11. Prepare substrate. Each sample should have 1x105 cpm of labeled IPS in ~10 nmols of cold IPS and 10 nmole PS. Dry lipids down under nitrogen, then resuspend in appropriate amount of assay buffer (50 ~100 mM Tris HCl, pH 7.4, 5mM MgCl2, 5mM DTT and 0.1% Triton X-100.

12. Vortex vigorously and sonicate if needed to completely solubolize the lipids.

13. To all tubes add 100 µl of assay buffer.

14. Mix samples gently and incubate for 30 minutes at 30°C.

15. Add 1.5 ml of chloroform:methanol (2:1) to stop the reaction and vortex.

16. Add 0.2 ml of 1 % perchloric acid to break the phases and vortex (For the separation of phases in assays using IPS, 0.2 ml of 1% perchloric acid was used instead of water. For M(IP)2C, lipid extraction by the method of Folch et al. was repeated three times.).

17. Centrifuge at 3,000 rpm for 5 minutes in a table top centrifuge to clarify the phases.

18. Take 400 µl of the upper phase and do scintillation counting. Remember to count 100 µl of substrate for total activity.

Ref. J Biol Chem 2000, 275(50):39793-8