Tracedrawer software package

Protein-protein interactions were measured using an OpenSPR LSPR biosensor (Nicoya Life Science, Kitchener, Canada), as described previously^{71 (link)}. In brief, 200 mL (50 μg/mL) of GST-GmRR11d was immobilized on a COOH sensor chip (Nicoya #SEN-AU-10012-COOH) at a flow rate of 20 mL/min in 1× PBS buffer (pH 7.4, RNase-free) and 0.1% (v/v) Tween 20. Free activated carboxyl groups were deactivated with the addition of 200 mL of blocking buffer (Nicoya). The immobilized protein was washed with running buffer (1× HEPES pH 7.4, 0.005% Tween 20) to reach a stable baseline. Buffer matched recombinant MBP-GmNSP1a (25 nM, 50 nM, 100 nM, 200 nM, 400 nM, 800 nM) or MBP-GmNSP2a (0.2 μM, 0.4 μM, 0.8 μM, 1.6 μM, 3.2 μM) was injected into the flow cell at a rate of 100 μL/min. Following a 5 min interaction time, the dissociation was recorded for an additional 7 min. Kinetic binding analysis was performed with the TraceDrawer software package (Ridgeview Instruments, Uppsala, Sweden). Sensorgram traces were fit to a 1:1 Langmuir model to derive affinity (Kd) constants.

Binding kinetics of purified peaks 2 and 3 samples to the hAPN were measured by surface plasmon resonance (OpenSPR, Nicoyalife), as described previously^{56 (link)–58 (link)}. In brief, the hAPN (200 µl, 5 µg) was immobilized on the OpenSPR™ COOH Sensor Chip (Nicoya # SEN-AU-100-12-COOH) at a flow rate of 20 µl/min in HBS-EP+ Buffer (GE Healthcare). Free activated carboxyl groups were deactivated with the addition of 100 µl blocking buffer (Nicoya). Then, the immobilized protein was washed with HBS-EP+ Buffer. After achieving a stable baseline, the running buffer was injected for blank measurement followed by successive injections of buffer matched peak 2 (0, 6.25, 12.5, 25, and 50 nM) and peak 3 (0, 3.13, 6.25, 12.5 and 25 nM) at 20 µl /min, and the binding time was 240 s and the natural dissociation 180 s was carried out. Response unit (RU) values were measured at 298 K. Binding kinetic parameters were obtained by fitting the curve to a one-to-one binding model using the TraceDrawer software package (Ridgeview Instruments, Uppsala, Sweden). All injections were carried out in duplicate and gave essentially identical results. Only one of the duplicates is shown.