Ribonucleotide reductase activity assay

Indicated cell lysates (500 µg) were incubated with the reaction mixture contained 50 mM Hepes buffer pH 7.2,10 mM DTT, 20 μM FeCl₃, 5 mM magnesium acetate, 50 μM CDP, 300 μM C¹⁴-CDP and 2 mM ATP at 37°C for 1 hr in a final volume of 50 µl. After incubation, 4 μl of 10 M perchloric acid was added to stop the reaction. After centrifugation, the supernatant was transferred to a new tube and boiled for 20 min. The supernatant containing the formed C¹⁴-dCDP and substrate C¹⁴-CDP were spotted on TLC (thin-layer chromatography) plate and separated by TLC. TLC plates were exposed to X-ray film and the dots of C¹⁴-CDP and C¹⁴-dCDP were quantified by Image J. The RNR activity was calculated as C¹⁴-dCDP/ (C¹⁴ -CDP+C¹⁴-dCDP).