CAT ASSAY[TLC METHOD]
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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 dH2 O (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.
