Infinite f200 m200 plate reader

APX1 activity was measured spectrophotometrically according to a previous report with a simple modification (Nakano and Asada, 1987 ; Jiang et al., 2016 (link)). In brief, a reaction mixture containing 275 μl buffer (KH₂PO₄/K₂HPO₄ [pH 7.0]), 10 μl 20 mM AsA, and 10 μl APX1 protein sample was added to a 96-well plate, and 5 μl 0.2 M H₂O₂ was added to start the reaction. The oxidation of AsA was recorded as a decrease in A₂₉₀ using a TECAN Infinite F200/M200 plate reader (TECAN, Switzerland) at 25°C. The reaction rates were linear for at least 3 min and were corrected for AsA auto-oxidation in the presence of H₂O₂. The APX activity was calculated using an extinction coefficient of 2.8 mM⁻¹ cm⁻¹ for AsA, and the enzyme activity unit was defined as μmol AsA mg protein⁻¹ min⁻¹.