Ref. Seed and Sheen, Gene 67, 271-277 (1988)
Normal reaction is Chloramphenicol + Acetyl-CoA -> Acetylchloramphenicol
Assay uses butyryl-CoA (more hydrophobic)
14C-Cm+ Bu-CoA->Bu-14C-Cm Km (Cm) 10 µM
Km (Ac-CoA) 50µM
The 14C- Butyrylated Cm is separated from 14C Cm by phase extraction.
ASSAY
Make up cocktail, keep on ice; does 10 assays
0.25 M Tris-Cl pH 8.0 0.7 ml (0.07/assay)
14C Cm (57µCi/µmol; 25µCi/ml) 0.1 ml (10µl/assay)
Bu CoA (5mg/ml) 0.2 ml (20µM/assay)
Total 1.0 ml (100µl/assay)
for assays longer than 1 hr, may need to add extra Bu-CoA to reactions at 1 hr.
Reaction (set up on ice)
Extract (50µl of 200 total) 50 µl
Cocktail 100 µl
Total 150 µl
Incubate 37oC, 30 min to 5 hr to overnight (1 hr standard)
To samples at room temp. add 300µl (2vols.) xylene; vortex hard 30 sec
microcentrifuge 3 mins; remove 270 µl (care)
add 150µl TE, vortex hard , spin 1 min, remove all xylene
add 150µl TE, vortex hard, spin 1 min, remove 250µl xylene to a scintillation vial. Add 4 ml scint, count use
1 CAT unit acetylates 1 µmol chloramphenicol/min 37oC
