Fig. 4. Assays for phosphatase activity of recombinant wild-type and mutant LMW-PTP.

Recombinant wild-type and substrate-trapping mutant proteins were assayed using the SensoLyte pNPP Colorimetric Protein Phosphatase Assay Kit (AnaSpec, Fremont, CA, USA). (A). Phosphatase activity of recombinant wild-type LMW-PTP with or without phosphatase inhibitor added. 10 to 200 ng of recombinant wild-type LMW-PTP was assayed at the 60 minute time point. To test inhibition, HALT™ Phosphatase Inhibitor Cocktail (Thermo Fisher Scientific, Waltham, MA, USA) was added at 1X to reactions with 50 to 200 ng wild-type LMW-PTP. Values were plotted as ng of recombinant wild-type LMW-PTP protein vs absorbance at 405 nm using Microsoft Excel. The trendline slopes and the R² values are also shown. Wild-type LMW-PTP phosphatase activity was inhibited by an average of 92.5 ± 1.0 % (± SD) for each sample. (B). Comparison of wild-type and mutant LMW-PTP phosphatase activity. 100 ng of either recombinant wild-type or substrate-trapping mutant LMW-PTP protein was assayed for 120 minutes, with A₄₀₅ measured every five minutes. Data was plotted as A₄₀₅ vs time (minutes) using Microsoft Excel. The slopes of the trendlines and the R² values are also shown.