4.7. TRAP Activity Assay

The cell lysates were extracted with passive lysis buffer (Polyplus transfection, New York, NY, United States) for 15 min at room temperature. TRAP activity was measured by mixing the cell lysate with a substrate mixture at 37 °C for 1 h and shielding it from light. Then, the reaction was stopped by adding 50 μL of 3 M NaOH into each well of the 96-well plate, and absorbance was measured at 405 nm. The substrate mixture consisted of a p-nitrophenyl phosphate (pNPP) substrate solution (610 mM pNPP in a buffer containing 0.1 M glycine (pH 10.4), 1 mM MgCl₂, and 1 mM ZnCl₂) and tartrate acid substrate buffer (89.3 mM of tartrate solution and 0.1 M of acetate solution in deionized water). The volume ratio of pNPP substrate solution to tartrate acid substrate buffer was 100:1. The tartrate acid substrate buffer was pre-heated to 37 °C before adding to the pNPP substrate solution.