SPR for Binding Studies.

The binding affinity between mNecl-4 and mNecl-1 Ig1 was analyzed on a Biacore 3000 machine with CM5 chips (GE Healthcare) at room temperature (25 °C). For the SPR measurements, all proteins were purified by gel filtration using a Superdex 75 column (GE Healthcare) and diluted in Hepes buffer consisting of 20 mM Hepes (pH 7.5), 150 mM NaCl, and 0.005% (vol/vol) Tween-20. Necl-4 (WT) and Necl-1 Ig1 (WT) were diluted to 20 μg/mL and immobilized on a CM5 chip using a standard amine coupling method.

The analytes were diluted in running buffer to a series of concentrations and injected at 30 µL per min for 60 s, after which they were subjected to a 120-s dissociation phase. The binding signals returned to baseline after the dissociation phase finished. The binding affinity (K_D) was analyzed with BIAevaluation Version 4.1 using the 1:1 Langmuir binding model. These protein concentration ranges were obtained using a twofold dilution series. The analyses were performed in duplicate to increase the concentration. The collected data and the final statistics are summarized in Table 3.