Chloramphenicol acetyltransferase (CAT) assay

The CAT assays were performed as before (30). Briefly, 11 μl of diluted or undiluted lysate was mixed with 15.5 μl of reaction mix (500 mM Tris–HCl, pH-8.0, 5 nmol chloramphenicol, 0.05 μCi ¹⁴C-chloramphenicol (Sp. activity 57.8 mCi mmol; ARC) and the reaction was started with addition of 3.5 μl of acetyl CoA (3.5 mg/ml). After 20 min of incubation at 37°C, the reaction was stopped using 300 μl ethyl acetate. After centrifugation, 200 μl of the supernatant was vacuum dried and spotted on a silica gel 60 plate (Merck). Thin layer chromatography (TLC) was performed using mobile phase consisting of chloroform and methanol in a 95:5 (v/v) ratio. The TLC plate was scanned post 6 h exposure using a Bio-Image Analyzer (FLA5000, Fujifilm). The densitometric analysis for quantification was done using Multi Gauge V2.3.