Biacore t100

The SPR experiments were carried out on a BIAcore T100 (GE Healthcare, Sweden) using CM5 sensor chips at 298 K. AAL2 protein solution was diluted to a concentration of 20μg/ml with fixing buffer (pH 5.0) and subsequently immobilized by amine coupling onto the chips. The immobilized level of AAL2 reached 3000 response units. Flow path 1 was used as an activated blank control for subtraction. Interaction experiments were performed in a running buffer of 50mM Tris-HCl pH 7.5, 150mM NaCl, 0.005% Tween 20 at a flow rate of 30μl/min. The ligands in sample buffer were employed as the flowing phase respectively. Each interaction experiment was carried out with gradient concentrations from low to high programmed to inject at a 30μl/min flow rate under a sensorgram. Binding curves were corrected, aligned and fitted kinetically using the BIAcore T100 evaluation program (GE Healthcare).

SPR experiments were carried out using a BIAcore T100 instrument (GE Healthcare). CT^C1 were immobilized on CM4 chips according to the standard amine coupling protocol. Briefly, carboxymethyl groups on the chip surface were activated with a 1:1 mixture of N-ethyl-N-(dimethyaminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS). Proteins were diluted in 20 mM HEPES, pH 6.5 and injected over the activated chip surface. The unbound chip surface was blocked using ethanolamine. Proteins were immobilized to approximately 200 response units. Different concentrations of analytes were injected over the immobilized chips at a flow rate of 30 μl/min. The sensor surface was regenerated by injection of 30 μl of 0.1% SDS and 60 μl of 500 mM NaCl. Reference responses were subtracted from flow cells for each analyte injection using BiaEvaluation software. The resulting sensorgrams were anaylsed to determine the kinetic parameters. Raw data shows a rise in signal associated with binding followed by a diminished signal after application of wash buffer.

SPR experiments were performed using a Biacore^® T100 instrument (GE Healthcare, Uppsala, Sweden). All binding analyses were performed at 25 °C using HBS-EP + (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.005% Tween-20) as the running buffer. Experiments were executed following the previously described SPR protocol.^{52 (link)} Briefly, RBD protein was immobilized on the sensor chip CM5-type surface raising a final immobilization level of ~600 Resonance Units (RU). Serial dilutions of 35B5 were injected in concentration from 10 to 0.625 nM. Serial dilutions of 32C7 were injected in concentration from 120 to 7.5 nM. For the competitive binding assays, the first sample flew over the chip at a rate of 20 μl/min for 120 s, then the second sample was injected at the same rate for another 200 s. The response units were recorded at room temperature and analyzed using the same software as mentioned above.

Surface plasmon resonance measurements using a BIAcore T100 (GE Healthcare Life Sciences) were performed essentially as described by Karlsson et al.^{50 (link)}. In brief, antibodies were coupled to CM3 sensorchips using an amine coupling kit (GE Healthcare Life Sciences) at a surface density of approximately 500 RU and a serial dilution (between 6.25 nM and 25 pM) of glycosylated human PAI-1 (and rat PAI-1 (both Molecular Innovations) in HBS-EP + (GE Healthcare Life Sciences) was passed over the sensorchip surface. The resulting sensorgrams were evaluated using the BIAcore T100 Evaluation software (version 2.0.3, GE Healthcare Life Sciences) to provide kinetic data.

The surface plasmon resonance (SPR) measurements were performed on a Biacore T100 (GE Healthcare) at 25°C. Approximately 30 response units (RU) of 3′-biotynilated S1 primer was immobilized on the flow cells of a streptavidin chip previously equilibrated in running buffer (25 mM Tris–HCl (pH 7.4), 140 mM NaCl, 5 mM EDTA, 2 mM DTT, 0.1 mg/ml bovine serum albumin, 0.005% surfactant P20). To prepare double-stranded DNA fragments carrying the putative LexA or gp6 target sequences within the P3 promoter, complementary primers (denoted as primer name_u and primer name_d) (Supplementary Table S1) were annealed using a temperature gradient as described in (12 (link)). The resulting DNA probes were 36–89 bp long and carried a 15 nt overhang complementary to the SPR chip-immobilized S1 primer at their 5’-end. A total of 30–100 RU of each DNA probe was immobilized onto each flow cell 2 at 2 μl/min, as detailed in the figure legends. The interaction between chip-immobilized DNAs and gp6, gp7, and LexA in different combinations was studied by injecting solutions at the desired protein concentration in running buffer at a rate of 100 μl/min. Regeneration of the sensor surface was performed with 50 mM NaOH for 10 s. SPR experiments were performed at the Infrastructural Centre for Analysis of Molecular Interactions at the Department of Biology, University of Ljubljana.

Mouse IgG2b Fc and IgG2c Fc were immobilized on a CM5 sensor chip using amine coupling on a Biacore T100 (GE Healthcare). Lane 1 was used for a control with no protein immobilized. Carboxymethyl dextran was activated by flowing over a 1:1 mixture of 0.4 M 1-ethyl-3(3-dimethylaminopropyl)carbodiimide and 0.1M N-hydroxy-succinimide for 7 min at 5 μL/min. Mouse IgG samples were diluted to a concentration of 1 μg/mL in 10 mM sodium acetate, pH 5.0, and flowed over the CM5 chip for 10 min at 5 μL/min. Unreacted sites were blocked with 1 M ethanolamine, pH 8.5, for 7 min. Typical immobilization response units varied from 400–1000. The CM5 chip used a coupling buffer containing 20 mM MOPS, 100 mM sodium chloride, and 0.05% P20 surfactant (GE Healthcare). The binding analysis buffer was identical but contained 1 μM bovine serum albumin. All experiments were carried out at 25°C. The CM5 chip was regenerated between runs with 100 mM glycine, pH 3.0, washes for 30 s to remove any bound receptor. Dissociation constants and rates were determined by averaging the values from at least two independent experiments. The error for this mean was determined by propagating the errors associated with the least-squares fit of each individual value. Differences in values were assessed by comparing the propagated error associated with each mean value.