Biaevaluation software 4

Kinetic titration series were performed in the interaction buffer (PBS with 0.5% Polysorbat 20, 0.1% BSA and 10% NSB reducer from GE Healthcare). 2.4 µM scFv IC16 was diluted four times with a dilution factor of two. To measure the interaction between Aß(1–42) and scFv IC16, the association and dissociation times were 270 and 90 s, respectively, for every analyte concentration. Further steps are comparable with “Parallel sensor kinetics of GB1 with biolayer interferometry” (Data Analysis software: 8.0.0.35). The sensorgrams with the concentrations: 0.24, 0.12, 0.06, 0.03 and 0.015 µM were fitted with the BiaEvaluation software 4.1 from Biacore using a 1∶1 binding model without RI-term.

The interaction of CR with curli fibers was analyzed by Surface Plasmon Resonance using a BIACORE 3000 biosensor system (GE Healthcare). Analyte (CR) dilutions were performed in HBS-EP (10 mM HEPES, pH 7.4, 150 mM NaCl, 3mM EDTA, and 0.005% v/v Surfactant P20) running buffer. Curli were immobilized onto a CM5 biosensor chip by amine coupling chemistry using N-hydroxysuccinimide (NHS) and N’-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC). To investigate binding of CR, the diluted analyte was injected over the curli fiber surface. The experiments were performed at 25°C using a flow rate of 50 L/min. For each experiment, at least 5 different concentrations of CR were injected over each experimental and control flow cell for 150 s. Dissociation was allowed to occur at the same flow rate for 180 s in Running Buffer alone. The surface was regenerated after each injection of analyte using two 18 s pulses of 50% DMSO/ 10mM NaOH which led to complete dissociation of the residually bound CR. All data were corrected for non-specific binding by subtracting the signal measured in a control cell lacking immobilized ligand. Both data processing and kinetic fitting were performed using Scrubber software, version 2 (BioLogic Software, Pty., Australia) or BIAevaluation software 4.1 (Biacore).

To measure the affinity between IgG and GB1, the association and dissociation phases were recorded for 270 and 90 s, respectively, for every analyte concentration (same concentrations as described in “Parallel sensor kinetics of scFv IC16 with biolayer interferometry”). Five sensors recorded the kinetic titration series, whereas one sensor recorded the buffer reference signal and six sensors were used as sensor reference. The other steps are comparable with section “Kinetic titration series of GB1 with biolayer interferometry” (Data Analysis software: 8.0.0.35). The sensorgrams with the concentrations: 0.24, 0.12, 0.06, 0.03 and 0.015 µM were fitted with the BiaEvaluation software 4.1 from Biacore with a 1∶1 kinetic titration series model without RI-term [4] (link).