CAT ASSAY (liquid phase)

 

 

<center> <font>CAT ASSAY<br /> (liquid phase)</font></center>

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 @ 37C
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 @ 37C.
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)