CAT?ASSAY[TLC?METHOD]

CAT ASSAY[TLC METHOD]

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'.

7. Resuspend in 100 µl 0.25M Tris pH 7.8

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 40 µl of supernatant to each.

12. In separate tube mix:

15 µl dH₂O (times number of samples + 1)

70 µl 1MTris Ph 8.0 (times number of samples + 1)

20 µl 4 mM Acetyl CoA (times number of samples + 1) and

5 µl 14 C CAM (times number of samples).

13. Add 0.5 ml Ethyl Acetate to each tube.

14. Vortex 10" each tube & then mix by inverting for another 30".

15. Spin @ 14K RPM for 30"-1'.

16. Remove 450 µl of organic (upper) phase.

17. Speed vac for 15'.

18. Resuspend in 30 µl Ethyl Acetate.

19. Spot on silica gel TLC (in 5 µl amounts).

20. Develop TLC in 200 ml Chloroform/Methyl-OH (95:5) until solution is almost at the top (app. 1 hr.).

21. Dry & expose against film ON.