The cascade reaction of IRED15 and NfsA was performed in a sequential manner. First, a reaction was prepared as in the IRED screening described above, but using 40 µM IRED15. After 4, 24 and 48 hours, samples of 100 µL were taken and analyzed as described above. After analyzing the 48 hour time samples, 10 µM NfsA was added and incubation was continued for a further three hours. A sample of 100 µL was analyzed using the method described above.