Fig. 5.

NbD7 forms a more stable complex with Etf-1 than does NbD3. (A) To obtain the elution profile of individual protein, recombinant protein Etf-1 (blue), NbD7 (red), or NbD3 (green), was individually separated by size exclusion chromatography. Fractions (numbered and shown in red at the bottom) corresponding to the elution peaks indicated by the absorbance at 280 nm were collected, and proteins in each fraction were subjected to SDS-PAGE and Coomassie blue staining. (B) To determine stability of binding complexes of recombinant Etf-1 + NbD7 (red) or NbD3 (green) in solution, each mixture was subjected to size exclusion chromatography. Coomassie blue staining shows the coelution of the stable complex of Etf-1 and NbD7 (red box), but coelution of Etf-1 and NbD3 was undetectable (green box). MW, molecular mass marker (indicated in kDa; A and B). (C) Binding affinity of D7 and D3 to Etf-1 was determined by OpenSPR. A series of five dilutions of Nbs was used to test their binding against biotinylated Etf-1 immobilized on a sensor chip. Colored lines are signals detected by OpenSPR, thin black lines are fitted models generated by the TraceDrawer software.