Biacore s200

SPR experiments were carried out using a Biacore S200 instrument (Biacore, GE Healthcare, Boston, MA, USA) at 25°C. Summarily, ILK (ligand) was first immobilized to the activated CM5 sensor chip by amine coupling with pH 5.0 and sodium acetate concentration of 46 μg/mL. The nonreaction group was then blocked by injection of ethanolamine hydrochloride 1 M (35 μL). The drug (analyte) was dissolved in 100% DMSO (to an initial concentration of 10 mM) and diluted to 200 μM (5% final DMSO concentration) with 20 mM HEPES, 150 mM NaCl, and 0.005% surfactant P20 buffer (HBSP). It was further diluted with HBSP + 5% DMSO (HBSP5%D). We then confirmed concentrations of compounds based on the solubility and the test results from sensor chips of samples to be measured. An increase in the RU value from the baseline indicates that formation is complex and that the plateau region represents a steady-state phase of interaction (RUeq), whereas a decrease in RU after 100 s indicates separation of the analyte from immobilized ILK after injection of HBSP5%D buffer. Finally, we performed sensorgram analysis using the Biacore S200 evaluation software.

Real-time biomolecular interactions between integrin αvβ3 and 24HC or 25HC were investigated by SPR using a Biacore S200 instrument (Cytiva). To create an appropriate interaction surface (R_max ~ 25), purified human αvβ3 Integrin (purchased from Yo proteins AB, Huddinge, Sweden) was covalently immobilized on a flow cell of CM5 chip (carboxy-methylated dextran coated) in 10 mM sodium acetate buffer, pH 4.0, using EDC/NHS amine coupling chemistry at 25 °C. The unused dextran surface was then inactivated by injecting 1 M ethanolamine, pH 8.5. The corresponding blank control flow cell was then activated and inactivated without the protein for background binding correction. For kinetic analysis, increasing concentrations of 24HC (or 25HC), as indicated in the figures (Fig. 6), in the running buffer PBS-P + (20 mM phosphate, 2.7 mM KCl, 137 mM NaCl, 0.05% polysorbate 20, pH 7.4) with 1% DMSO were injected at a flow rate of 30 μL/min for 180 s. Following dissociation for 600 s, the chip surface was regenerated with the running buffer. The SPR sensorgrams were plotted and quantitatively evaluated to determine the affinity constant (K_D) using the Biacore S200 Evaluation Software (Cytiva) and the steady-state equilibrium binding model.

PSMD2 was biotinylated via an Avi tag on the N terminus and immobilized on an SA Series S SPR chip (Cytiva) for measurements on a Biacore S200 (Cytiva). All four flow cells were blocked with saturating amounts of PEG-biotin (Thermo) after immobilization to prevent nonspecific binding. Experiments were run in 25 mM HEPES (pH 7.5), 100 mM NaCl, 90 mM KCl, 2% glycerol, 0.005% Tween-20 and 1% DMSO at 10 °C. Ligands were diluted three times in either a six- or ninefold dilution series and injected at 50 µl min⁻¹ using single-cycle kinetics with sufficient off rates to determine kinetic rate constants of dissociation. A five-point solvent correction was run before and after the samples to account for DMSO-based bulk shifts. Binding kinetics were fit using a 1:1 binding model in Biacore BiaEvaluation (Cytiva) with an added parameter to account for drift over very long off rates.

Measurements were performed on a Biacore S200 instrument (Cytiva Europe GmbH, Freiburg, Germany). rHbl B’ was diluted to a final concentration of 150 nM in 10 mM sodium acetate, pH 4.8, and chemically immobilized (amine coupling, 350 RU bound) onto series S sensor chip CM5 (Cytiva Europe GmbH, Freiburg, Germany). rHbl L1, L2, and B protein samples were diluted in a running buffer (PBS, 1 mM DTT and 0.005% Tween 20) to the final concentration of 7.8125 nM, 15.625 nM, 31.25 nM, 62.5 nM, 125 nM, 250 nM, 500 nM, 1 µM, and 2 µM and injected over the sensor chip surface at 30 µl/min at 20°C from the lowest to the highest concentration. Injection of 250 nM protein concentration was always performed in duplicate within each experiment. For the sensor chip regeneration after each injection, 1 M MgCl₂ solution has been used for rHbl L1 and 0.5 M NaCl solution for rHbl L2 and B. In order to subtract any background noise from each experiment, all samples were also run over an unmodified sensor chip surface. Data analysis was performed with Biacore S200 evaluation software v1.1. For each measurement, the equilibrium dissociation constant (K_D) was calculated using a 1:1 binding model. The K_Ds from three experiments were used to calculate the mean values of these variables and the SEM.

Surface plasmon resonance
(SPR) ligand interactions assays were performed on a Biacore S200
(Cytiva Life Sciences) at 2 °C using multi-cycle settings. Biotinylated
avidin-MAPK14 protein (MRC-Reagents, Dundee) was immobilized onto
a Streptavidin surface chip, through injection of 50 μg/mL MAPK14
in dimethyl sulfoxide (DMSO)-free SPR running buffer (20 mM HEPES,
150 mM NaCl, 0.1 mM EGTA, 0.5 mM tris(2-carboxyethyl)phosphine (TCEP),
0.01% Tween-20, pH 7.4) over the active flow cell eliciting final
captured response units (RUs) of 7719 RUs. The inhibitor analytes
(20 mM HEPES, 150 mM NaCl, 0.1 mM EGTA, 0.5 mM TCEP, 0.01% Tween-20,
pH 7.4, 1% DMSO) were then injected over both control and active surfaces
for 90 s at 30 μL/min before being allowed to dissociate for
600 s over 10 concentration series to record dose responses: 0.05–333.33
nM. A solvent correction was applied to the data collection, and an
8-point DMSO solvent correction was applied. Responses were analyzed
using Biacore Evaluation Software (Cytiva Life Sciences) using affinity
fit to determine the K_d. Data are representative
of three technical replicates.