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. 2016 Apr 5;6:23582. doi: 10.1038/srep23582

Figure 2. Biochemical analysis of direct interactions of purified H. pylori TlpD and AcnB or TlpD and KatA proteins using biolayer interferometry.

Figure 2

Recombinantly purified TlpD was tested for direct interactions with purified H. pylori AcnB or KatA using biolayer interferometry. While one purified protein (ligand) was coupled to the sensor surface via a hexa-histidine tag or free amine groups (see Supplementary Methods), the second protein (analyte) was applied as a solute in assay buffer. Sensors were dipped into different analyte solutions for 1,200 sec (A) or 1,800 sec (B), before dissociation was monitored in assay buffer. (A) TlpD-AcnB interaction: ligand Hisx6-TlpD (purified from E. coli) was immobilised to the sensor surface, while analyte H. pylori AcnB (AcnB-V5, purified from H. pylori) was applied in assay buffer (Methods and Supplementary Methods) at four different concentrations (26.4 nM, 52.8  nM, 158.5 nM and 317.0 nM). (B) TlpD-KatA interaction: immobilised H. pylori KatA (recombinant, tag-free, purified from E. coli) was coupled on the sensor surface via free amine groups, while analyte TlpD-V5 (purified from H. pylori) was supplied diluted in assay buffer at 200 nM, 400 nM and 2,000 nM. Values derived from a ligand-coupled sensor that was dipped in assay buffer only over the full time course of the interaction assay were subtracted as the baseline from the interaction curves.