PAGE-Based Measurements of nsP2 Protease Activity.

In this protocol, we describe a PAGE-based assay to evaluate the steady-state kinetics of the nsP2 protease. Using a recombinant GFP-TRX fusion substrate containing the nsP3/4 cleavage site, proteolytic activity is monitored through the appearance of cleavage products resolved by SDS-PAGE. Detection is achieved by Coomassie blue staining or GFP fluorescence emission, followed by densitometric analysis to quantify product formation and calculate initial reaction rates. The protocol provides a robust and accessible method for comparing enzyme activity under varying conditions, and is suitable for studies of substrate specificity, inhibitor screening, and the mechanistic characterization of protease function.