Preparation of 3[H]inositol labeled IPS from yeast cells (Silvia, Yasuo)

Strains; Deletion mutant of ISC1, MIPC synthase and M(IP)2C synthase

1. Day1, inoculate 5 ml of YPD with a single colony, and grow cells at 30 ûC overnight.

2. Day2, dilute the culture to get the cells in the mid-log stage of growth next morning and grow cells at 30 ûC overnight.

3. Day3, adjust cell density (for example, 3 x 107 cells/ml, OD600=0.6~0.8) and spin down cells at 3,000 rpm X 5 min.

4. Remove the medium and wash cells with water.

5. Add SC-inositol medium add 3 [H]inositol (10~20 µCi/ml) to tube, divide 1 ml into each tube and continue to incubate at 30 ûC in the shaker.

6. After incubation, transfer cells to a glass tube, spin down the cells , aspirate the medium and wash the cells with water.

7. Add extraction buffer (ethanol : water : ether : pyridine : concentrated (14.8 N) NH4OH=15 : 5 : 5 : 1 : 0.08) and vortex.

8. Incubate at 60 ûC for 15 min and spin down at 3,000 rpm X 5 min.

9. Transfer the supernatant to new glass tube.

10. Add 0.5 ml of extraction buffer and repeat steps 8-10.

11. Combine the supernatant and dry down.

12. Add 0.5 ml 20 % monometylamine to each dried down lipid sample and vortex to resusuped.

13. Incubate 52 ûC for 45 min.

14. Spin down and transfer the supernatant to new glass tube.

15. Repeat steps 13-15.

16. Combine the supernatant and dry down.

17. Add CH3Cl : MeOH : water= 16 : 16 : 5 to dissolve lipids and vortex.

18. Apply samples onto TLC plates.

19. Develop TLC in the following solevent system. CH3Cl ; MeOH : 4.2 N NH4OH= 9 : 7 : 2

20. Spray ENHANCE spray to the edge on theTLC plate and put &endash;80 Cû.

21. After 2 days, develop TLC plate.

22. Scrape IPS bands and extract lipids using CH3Cl : MeOH : water= 16 : 16 : 5 three times.

23. Dry down and resuspend CH3Cl : MeOH : water= 16 : 16 : 5.

24. Measure total phosphate to decide the conc.

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