Ethanolamine hydrochloride naoh

SPR analysis were performed by Creative Biolabs using a Biacore T200 instrument (Cytiva). NT5C2 D52N R367Q recombinant protein was directly immobilized on the CM5 chip using an amine coupling kit (Cytiva). Before immobilization, the CM5 sensor surface was activated using a mixture of 400 mM 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC) and 100 mM N-hydroxysuccinimide (NHS). Then, 50 μg ml⁻¹ of NT5C2 protein in immobilization buffer (10 mM NaAc (pH 4.0)) was then injected into Fc2 sample channel at a flow rate of 10 μL min^-1. The amount of ligand immobilized was about 15,000 RU. The chip was deactivated by 1 M Ethanolamine hydrochloride-NaOH (GE Healthcare Life Sciences) at a flow rate of 10 μL/min for 420 s. The reference Fc1 channel underwent similar procedures but without injecting the ligand. The analyte (CRCD2) was serially diluted with the running buffer, in the absence or the presence of 500 μM IMP, to give a concentration of 62.5, 31.25, 15.625, 7.813, 3.906, 1.953, and 0 μM, respectively. Different concentrations of analytes were then injected into the Fc2-Fc1 of channels at a flow rate of 30 μL min⁻¹, with a contact time of 60 s, followed by a dissociation time of 90 s. Data analysis was performed on the Biacore T200 computer and with the Biacore T200 evaluation software, using the steady state affinity model.