Nadp nadph extraction buffer

To analyze the intracellular NADP⁺/NADPH ratio, E. coli were inoculated and cultured in LB medium for 16 h to reach stationary phase, and the cells were harvested by centrifugation (3300×g, 4 °C, 10 min). Cells were then washed with ice-cold phosphate-buffered saline, lysed with NADP⁺/NADPH extraction buffer (Biovision, Milpitas, CA, USA) in a microcentrifuge tube, and kept on ice for 10 min. The crude extracts were centrifuged at 15,000×g for 10 min to obtain the supernatant. The amount of NADP⁺/NADPH in the cells was measured using a NADP⁺/NADPH Quantitation Colorimetric Kit (Biovision). To measure the intracellular NADH/NAD⁺ ratio, a NAD/NADH-GloTM Assay Kit (Promega, WI, USA) was used. The culture medium was mixed with a DTAB solution for 5 min. To measure NADH, 0.4 N HCl was added to the reaction mixture. Samples were heated to 60 °C for 15 min and cooled to 25 °C before assay, following the manufacturer’s instructions.