Figure 5. Substrate serial dilution scheme and assay plate layout for Michaelis–Menten kinetics assay.

In step 1, a substrate serial dilution (1:1) is prepared at 5× final concentration. In step 2, 5 µL of each substrate solution (SS) is transferred into column 1, which serves as the background control and contains 20 µL of enzyme buffer (EB), and columns 2–4, which serve as the triplicate PTP reaction wells and contain 20 µL of enzyme solution (ES).