Figure 8

In vitro discovered nanobodies bind to purified SbMATE. SPR was performed and the data were double-reference subtracted as described in Fig. 6B, except purified nanobodies (Nbs) were injected at various concentrations, instead of ethidium. The calculated dissociation constants (K D) for the lower-affinity Nbs were (A) Nb 80–12.2 µM, (B) Nb 81–23.9 µM, (C) Nb 83–3.3 µM, and (D), Nb 93–26.1 µM. (E) Nb 86 and (F) Nb 94, bound with higher affinity, with K D’s in the nM range. To get more accurate K D values, a BSA-immobilized surface was used as the reference channel, instead of a blank surface. Calculated K D’s; Nb 86–173.57 ± 45.53 nM; Nb 94–99.53 ± 19.32 nM (n = 3 independent replicates, sensorgrams are from a single experiment).