Br 1000 50

The binding affinities and kinetics of IRCR201 for human c-Met and mouse c-Met were measured using a Biacore™ T100 (GE Healthcare Life Sciences, Uppsala, Sweden). Human c-Met, mouse c-Met, or BSA were immobilized on a CM5 sensor chip (GE Healthcare Life Sciences, BR100530, Uppsala, Sweden) using an Amine coupling kit (GE Healthcare Life Sciences, BR100050, Uppsala, Sweden). Different concentrations of purified IRCR201 were injected into immobilized human and mouse c-Met to determine K_D values. To obtain the kinetic and affinity constants of IRCR201 to human and mouse c-Met, Biacore™ T100 evaluation software (GE Healthcare Life Sciences, Uppsala, Sweden) was used.

Surface Plasmon Resonance (SPR) analysis was performed at 25 °C using the Biacore T200 (GE Healthcare). Purified sTREM2-Fc and Fc protein were immobilized onto Biacore CM5 (GE Healthcare, BR-1005-30) chip using amine coupling kit (GE Healthcare, BR-1000-50). oAβ_1–42, mAβ_1–42 or scAβ₄₂ was tested with a gradient concentration of 1.25 μM, 2.5 μM, 5.0 μM and 10.0 μM respectively.

Real-time surface plasmon resonance analysis was performed using the Reichert SPR Biosensor SR 7500 C instrument (Reichert Inc., NY, USA). Soluble recombinant human EPOR-Fc chimera protein (#963-ER, R&D Systems, Minneapolis, MN, USA) was covalently immobilized on a carboxymethylated dextran matrix-coated chip (BR-1005-39, Pharmacia Biosensor AB) by using an amine coupling kit (BR-1000-50, GE healthcare) according to the manufacturer's instructions. Each concentration (5, 2.5, and 1.25 μM) of the peptide flowed at 5 μl/min was tested in duplicate. After each binding cycle, the sensor chip was recharged by 20 μl/min injections of 25 mM acetic acid.

The kinetics of Ab10 and Gn glycoprotein binding were measured by surface plasmon resonance analysis, using a Biacore T200 instrument with sensor chip CM5, amine coupling kit, and his capture kit (28975001, 29149603, BR100050, 28995056; GE Healthcare). We followed the recommended manufacturer’s protocol for the procedures and conditions of reaction buffers, flow times, flow rates, and concentration of analytes. Briefly, anti-histidine antibody was immobilized on an activated CM5 chip, followed by a deactivation step. Then, histidine tagged Gn-Cκ was injected over the flow cells prior to antibody injection. For the association step, all of the Ab10 IgG₁ antibody in PBS at concentrations of two-fold increments ranging from 1.25 nM to 80 nM was injected for 3 min. For the dissociation step, PBS containing 0.005% of Tween20 was injected for 5 min. After each dissociation step, chip regeneration was performed.

Surface plasmon resonance (SPR) analysis was performed to determine the binding affinities of the compounds and recombinant NP using the Biacore T200 system (GE healthcare Bio-Sciences, Japan) as previously described [28 (link)]. Naproxen, previously reported to bind to NP [23 (link)], was used as a positive control. Purified NP was diluted to 20 μg/mL with phosphate buffer (pH 7.5) and immobilized on a CM5 sensor chip (GE Healthcare, BR-100, 530) using an amine coupling kit (GE Healthcare, BR-1000-50). A total of 8725 response units (RU) of NP were immobilized. Compounds were dissolved in running buffer (phosphate buffer saline, 0.05% Tween 20 and 5% DMSO) at concentrations that allowed total solubilization. Different concentrations of the compounds (NUD-1 3.9–62.5 μM) and naproxen (3.9–125 μM) were injected at a flow rate of 10 μL/min at 25°C for 60 sec. The DMSO concentration was maintained at 5% in all of the solutions. A blank sensor chip was used as a control.

Streptavidin was immobilized on the surface of a CM5 sensor chip (GE Healthcare, BR100399) using the amine coupling kit (GE Healthcare, BR100050). A solution containing EDC (7.5 mg mL⁻¹) and NHS (11.5 mg mL⁻¹) was injected into the flow cells to activate the carboxyl groups followed by a solution containing streptavidin (0.5 mg mL⁻¹) and finally ethanolamine (1 M) to block remaining activated carboxyl groups. All solutions were flowed through the channels for 7 min at a flow rate of 10 µL min⁻¹. Finally, biotinylated CA K158Cor biotinylated CA hexamer were immobilized on the surface by injection a 2.5 µM solution for 30 s at a flow rate of 30 µL min⁻¹. Flow channels modified with streptavidin were used as reference cells. CypA was buffer exchanged into SPR running buffer using a Zeba gel filtration spin column. CypA solutions at a range of concentrations (3.125–100 µM) were flowed through the cells (20 s at a flow rate of 100 µL min⁻¹) followed by a buffer wash (30 s at a flow rate of 100 µL min⁻¹) while measuring the SPR response at a frequency of 40 Hz.

Interactions between recombinant PA and compounds were evaluated by SPR using the Biacore T200 system (GE Healthcare UK Ltd, Buckinghamshire, UK), as described previously^{49 (link)} with some modifications. Briefly, we immobilised the anti-PA antibody on a CM5 sensor chip (GE Healthcare, BR-100530) using an amine coupling kit (GE Healthcare, BR-1000-50) and running buffer (10 mM HEPES pH 7.4, 150 mM NaCl and 0.1% Tween 20 [Sigma Aldrich]). The amount of immobilised antibodies was ~8,000 RU. Next, the recombinant PA protein were loaded at a flow rate of 30 μL/min for capture by the antibodies on the sensor chip. The amount of PA protein captured reached ~2,000 RU. Compounds were analysed using the running buffer containing 5% DMSO. Data were corrected by using the anti-PA antibody immobilised sensor chip as a control.