Preparation of cold 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 cells and grow cells at 30 ûC overnight.

3. Day3, remove the medium and wash cells with water.

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

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

7. Transfer the supernatant to new glass tube.

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

9. Combine the supernatant and dry down.

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

11. Incubate 52 ûC for 45 min.

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

13. Repeat steps 13-15.

14. Combine the supernatant and dry down.

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

16. Apply samples and labeled IPS as standards onto TLC plates.

17. 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