Streptavidin sa chip

SPR experiments were performed using BiaCore3000 (GE Healthcare). All proteins solutions were dialyzed against HEPES-buffer (sterile filtered and degassed) at 4°C for 2 h. TirM_EHEC was biotinylated (as described above) at 0.1 μg/ml and immobilised on a Streptavidin SA chip (GE Healthcare) at 150 response units (RU) at a flow rate of 10 μl/min in HEPES-buffer containing 0.005% (v/v) of the surfactant Polysorbate 20 (P20, GE Healthcare). To determine binding kinetics, dilutions of purified TD4-HlyA or Int280 (as indicated) were run at 30 μl/min in HEPES-buffer and sensograms were generated. Regeneration of TD4-HlyA was performed by sequential injections of 10 μl 10 mM glycine-HCl pH 1.7, 5 μl 5 mM NaOH and 10 μl 10 mM glycine-HCl pH 1.7. No regeneration was needed for Int280. Sensograms with different concentrations of analyte were overlaid, aligned and analysed with BIAevaluation 4.1 software (GE Healthcare) under assumption of the 1:1 Langmuir model and using both the simultaneous kinetics model and the steady-state equilibrium analysis [73 (link)].

Kinetic SPR measurements were made using a Biacore 3000 instrument (GE Healthcare) for DARPins pE59, pEM1, EpEM6, and EpEM7. The running buffer was 50 mM Tris (pH 7.4), 150 mM NaCl, 0.05 mM EDTA, and 0.005% Tween-20. Biotinylated ERK2 or pERK2 was immobilized on a streptavidin SA chip (GE Healthcare) to ~500 response units (RU). Interactions were determined by injecting varying concentrations of each DARPin at a flow rate of 30 μL/min for 5 min, after which off-rate measurements were made by flowing running buffer for 50 min. The signal of an uncoated reference cell was subtracted from the sensorgrams. Zero-concentration samples (Tris buffer) were also included in SPR experiments as a baseline for double referencing. Sensorgram data were evaluated by fitting the equilibrium binding responses to obtain affinity values using BIAevaluation software (GE Healthcare) and Prism software.