Binding affinity of small molecule to protein was measured on a BIAcore 8K instrument (GE Healthcare) by SPR assay. For a typical assay, purified hTNF‐α was dissolved at 30 µg mL−1 in an acetate buffer (pH 4.5), and coupled to a CM5 chip (GE Healthcare, 29‐1049‐88) following the manufacturer's instruction. The running buffer (PBS‐P, pH 7.4) contained 1% instead of 5% DMSO in order to keep the activity of hTNF‐α (Table S2 , Supporting Information). Chemicals were 1:2 serial diluted from 50 µm to 1.56 µm (final concentrations) and injected at a flow rate of 30 µL min−1 for 90 s for the association step followed by dissociation for an additional 90 s using the LWM multi‐cycle kinetics/affinity method provided by GE Healthcare. Solvent correction was carried out before and after each analysis with eight different concentrations of DMSO solution per cycle. The KD value was calculated using Evaluation Software (GE Healthcare).
Evaluation software
Manufactured by GE Healthcare
Evaluation Software is a suite of tools designed to analyze and assess the performance of laboratory equipment. The software provides a comprehensive set of features to measure and evaluate the functionality, efficiency, and accuracy of various laboratory instruments. It offers a standardized platform for data collection, analysis, and reporting, enabling users to make informed decisions about their laboratory equipment.
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2 protocols using evaluation software
1
SPR Assay for Protein-Ligand Binding
2
Kinetic Analysis of EcfG1 and EcfG2 Interactions with NepR1/NepR2
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EcfG1 and EcfG2 interaction kinetics with respect to immobilized NepR1 or NepR2 were measured using a BIAcore X100 device (GE Healthcare Life Sciences). Assays were performed at 30°C in TEDG buffer. NepR1 (12.1 RU) or NepR2 (36.4 RU) were immobilized on the surface of a CM5 chip using 10 mM acetate buffer pH 4.0 or 5 mM malate buffer pH 5.5 respectively, at 30°C with a contact time of 300 s, following the manufacturer's instructions. Serial two‐fold dilutions of EcfG1 and EcfG2 in TEDG buffer were injected into the system at a flow rate of 20 μl min−1 in concentrations ranging from 60 to 0.469 nM. Analyte contact time was enough to reach interaction equilibrium and dissociation time was 300 s. After each interaction cycle, the chip was regenerated by injection of 10 mM glycine‐HCl buffer pH 2.0. Data were fitted to a 1:1 interaction model using the evaluation software provided by the manufacturer (GE Healthcare Life Sciences). Reliability of the results was assessed according to U‐value <15 and χ2 < 5%Rmax. Interaction affinity was defined by the dissociation constant (KD) obtained for each NepR‐EcfG pair. At least three independent replicates were assayed for each pair.
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