CAT?ASSAY?(liquid?phase)

1. Transfer cells to a 15 ml tube.

2. Add 5 ml TBS- to flasks, shake & pour into tubes.

3. Spin down the cells @ 1K RPM for 5'.

4. Resuspend in 1 ml TBS-.

5. Transfer to 1.5 ml tubes.

6. Spin down @ 14K RPM for 2-3', remove supernatant.

7. Resuspend in 100 µl buffer I (250 mM Tris pH 7.8/5 mM EDTA).

8. Freeze-thaw 3x (10'freeze-10'thaw).

9. Spin down debris @ 14K RPM for 5'.

10. Transfer 100 µl supernatant to a new set of tubes.

11. Set new set of tubes and add 10 µl of supernatant and 50 µl buffer I in each.

12. Add 20 µl of 5mM chloramphenicol in buffer I(or 20 µl of buffer I for a blank).

13. Incubate 5 min @ 37℃

14. Make the following mix:

0.75 µl of 4mM acetyl CoA

2.0 µl 750 µM HCl (10X)

16.25 µl H₂ O

1.0 µl 3H acetyl-CoA

(Times number of samples)

15. Add 20 µl of mix into each tube (reaction & blank).

16. Incubate for 2 hrs @ 37℃.

17. Add 500 µl of toluene to each tube.

18. Vortex for 10 sec.

19. Spin down for 1 min.

20. Remove 400 µl into scintillation tubes.

21. Add 10 ml of scintillation liquid.

22. Count it for 2 min.

(total cpm's in reaction is 210000-230000)