Figure 9

Higher affinity Nbs inhibit ethidium binding to SbMATE. (A) SPR was performed as described in Fig. 6B, with the following modifications. 50 µM ethidium bromide was injected either directly on SbMATE alone, or following injections of 5 µM Nb 86 or Nb 94 without a subsequent regeneration step. The ethidium trace (top/black) represents double-referenced data as described in Fig. 6B, while the ethidium +Nb traces (Nb 86 – bottom/red; Nb 94 – middle/blue) were generated using a similar double-referencing method, except the respective Nb injections alone were used instead of a buffer injection for the blank subtraction (n = 3 independent replicates, sensorgram is from a single experiment). (B) The response value (in RUs) at the end of the association phase (i.e. at ~118 s) of the injections described in Fig. 9A were normalized, with the RU value for the ethidium-SbMATE trace set to 100%. Response RUs were reduced to 35.04 ± 13.39% for ethidium-Nb86-SbMATE and to 71.40 ± 13.91% for ethidium-Nb94-SbMATE (n = 3 independent replicates, mean ± SEM). (C) SPR was performed as described in Fig. 9A, except the non-specific hydrophobic control protein, BSA was immobilized on the sensor chip, instead of SbMATE. To obtain comparable ethidium signals, 2x higher levels of BSA than SbMATE were immobilized.