Bicinchoninic acid bca protein determination kit

Day 3 adult flies were transferred to cornmeal medium containing 10 mmol/L paraquat for CAT activity detection. After feeding for 24 h, approximately 20 mg flies were used for each replication and homogenized in 100 µl lysis buffer from the kit with 1% Triton. Supernatant of the homogenization was transferred to a new test tube for CAT analysis with the CAT activity assay kit (Beyotime Biotechnology, Shanghai, China). Briefly, under the catalysis of peroxidase, H2O2 residuals could oxidize the chromogenic substrate. Absorbance of the developed red color product, N-4-antipyryl-3-chloro-5-sulfonate-pbenzoquinonemonoimine, was detected at 520 nm on a uQuantMicroplate spectrophotometer (Biotek Instruments, Winooski, VT) after incubation for 15 min at 25°C. To calculate CAT enzyme activity, a standard curve was made using H2O2 as standard at the concentrations of 0, 0.625, 1.25, 2.5, 3.75, and 5 mM. Protein concentration of each sample was determined using a bicinchoninic acid (BCA) protein determination kit (Thermo Fisher Scientific, Waltham, MA) according to the manufacturer’s instructions.