Biacore t200 system

The binding affinities of C7-Fc and anti-CD239 mAbs were assayed by surface plasmon resonance analysis using a Biacore T200 system (GE Healthcare Bio-Sciences). The recombinant CD239 (Lu-Fc) was immobilized on a CM5 sensor chip (GE Healthcare Bio-Sciences). Various amounts of C7-Fc and anti-CD239 mAbs in 10 mM HEPES (pH 7.4), containing 150 mM NaCl, 3 mM EDTA, and 0.005% Tween 20, were injected into the CD239-immobilized flow cell with a flow rate of 50 μL/min. The K_d was determined by a nonlinear fitting method using the BIAevaluation software (GE Healthcare Bio-Sciences).

SPR experiments were performed in triplicate on a Biacore T200 system (GE Healthcare, Uppsala, Sweden), equilibrated at 25°C in PBS (pH 7.4) complemented with 0.01% Tween 20, using a CM5 sensor chip with a density of around 13,000 response units (RU; similar to measurement in picrograms per square millimeter) and anti-Strep antibody (C23.21) immobilized through amide bonds.
sE2_412-715 (5 µg/ml) was captured on the chip via its Strep tag at a density of 2,100 to 2,200 RU. The DAO5 or e137 Fabs (50 µg/ml) were then injected for 5 min, followed by buffer, DAO5, or e137. The Fab signal monitored on an anti-Strep reference surface was subtracted.

The immobilization of the PdgC PAS domain on the sensor surface was carried out as previously described (68 (link)). Briefly, PdgC was diluted in 10 mM sodium acetate (pH 5.5) and immobilized on a CM5 sensor chip using the amine coupling method by following the manufacturer’s protocol. SPR measurements were performed using a Biacore T200 system (GE Healthcare, Sweden).
PAH compounds were diluted in running buffer (phosphate-buffered saline [PBS] containing 5% DMSO) to a concentration of 50 μM. Running buffer alone (PBS containing 5% DMSO, without PAHs) served as the negative control. All solutions were then injected into the PdgC sensor surface for 120 s at a flow rate of 30 μl/min. Sensograms of these PAHs were recorded and analyzed. Analytes were injected through the reference and active channels at a flow rate of 30 μl/min. The association and dissociation times were both 120 s. Affinity fitting (determination of the affinity constant, K_D) was performed using Biacore T200 evaluation software with a 1:1 binding mode.

The SPR screening of inhibitors targeting SHCBP1–PLK1 was conducted using Biacore T200 system (GE Healthcare). All experiments were carried out with HBS-EP (10 mM HEPES pH 7.4, 150 mM NaCl, 3.4 mM EDTA, and 0.005% surfactant P20) as running buffer with a constant flow rate of 10 μL/min at 25 °C. PLK1, which was diluted with 10 mmol/L sodium acetate buffer (pH 5.0) to a final concentration of 10 μM, was immobilized on a CM5 Sensor Chip surface via covalent linkage to the N terminus of PLK1. Bindings of 40 compounds with PLK1 were performed by passing the molecules (100 μM) through the immobilized PLK1 at the flow rate of 10 µL/min. The association and dissociation time were 120 s, respectively. The compounds which response units >16 RU were screened and the kinetic analyses were performed. For kinetic analyses of the TFBG–PLK1 binding, TFBG was dissolved in the running buffer at different concentrations ranging from 0.78 to 12.5 μM and the kinetic analyses were performed based on the steady-state affinity fit model, according to the procedures described in the software manual.

SPR experiments were performed at 18 °C using a BIAcore T200 system (GE Healthcare) and an NTA sensor chip (GE Healthcare). Protein samples were prepared in 20 mm HEPES, pH 7.5, 500 mm NaCl, and all the measurements were recorded in the same buffer at a flow rate of 30 μl/min. A single cycle kinetics approach was used to study the interaction between PexRD54 and ATG8CL. The NTA chip was activated by injecting 10 μl of 0.5 mm NiCl₂ over flow cell 2, which was also used to immobilize His-tagged ATG8CL to a response level of 85 ± 2. Increasing concentrations of PexRD54 (20, 200, 600, 1000, and 2000 nm) were injected over flow cell 1 and 2 for 90 s. After the final injection, the dissociation was recorded for 300 s. Two startup cycles were run where the chip was activated and ATG8CL immobilized in the same manner, but buffer only was injected instead of PexRD54. This was subtracted to account for any dissociation of ATG8CL from the sensor chip. The sensor chip was regenerated by injecting 10 μl of 350 mm EDTA. The data were analyzed using BIAcore T200 BIAevaluation software (GE Healthcare) and then plotted with Microsoft Excel.

Further examination of the binding affinity (K_D) between prazosin and EphA2 was conducted via SPR analysis with a Biacore T-200 system (GE Healthcare). Briefly, human recombinant EphA2 (R&D) as the ligand was coated on the CM5 sensor chip, and an analyte gradient of prazosin from 0 to 25 μM was assayed in running buffer containing 0.05% Tween-20 and 1% dimethyl sulfoxide in phosphate-buffered saline at a flow rate of 10 μL/min with a contact time of 60 s.