CAT Assay

 

<center> <font>CAT Assay<br /> </font></center>
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

1. 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