The BIAcore T200 system was used to detect the binding capacity of thrombin and gallic acid. Ten millimole thrombin (Sigma, pH 5.0) was immobilized on the surface of CM5 sensor chip following standard amine coupling protocol. Different concentrations of gallic acid were diluted in 5% DMSO contained PBS-P (0.05% P20, pH7.4) and flow through the chip surface at a rate of 30 μL/min at 25 °C. In this assay, amine coupled chip without immobilized thrombin was set as the blank control. The association and dissociation kinetic processes of gallic acid on thrombin were continuously monitored for 4 and 10 min, respectively. Glycine-HCl (10 mmol/L, pH 2.0) treated chip could regenerate for reused several times. Compounds were detected using single-cycle kinetics mode in two-fold concentration series (1.17–37.50 µmol/L for gallic acid and 4.70–75.00 µmol/L for argatroban). The equilibrium constant (KD) was calculated using the ‘affinity’ module following 1:1 molecularity of reaction ratio between molecule and protein in Biacore T200 software.
T200 system
Manufactured by Cytiva
The T200 system is a label-free, analytical instrument designed for the characterization of biomolecular interactions. It utilizes Surface Plasmon Resonance (SPR) technology to measure the affinity and kinetics of binding events between molecules in real-time. The system provides quantitative data on association and dissociation rates, as well as equilibrium binding constants, without the need for labeling.
Automatically generated - may contain errors
Lab products found in correlation
Cm5 chip, by GE Healthcare (2 mentions)
Control software v 2, by Cytiva (2 mentions)
Thrombin, by Merck Group (1 mentions)
Br 1005 31, by Cytiva (1 mentions)
Hbs ep buffer, by Cytiva (1 mentions)
His capture kit, by Cytiva (1 mentions)
Biacore t200 evaluation software, by GE Healthcare (1 mentions)
Amine coupling kit, by GE Healthcare (1 mentions)
T200 evaluation software, by Cytiva (1 mentions)
Hbs ep buffer, by GE Healthcare (1 mentions)
C57bl 6 mice, by Charles River Laboratories (1 mentions)
Cm5 sensor chip, by Cytiva (1 mentions)
Biaevaluation software package, by GE Healthcare (1 mentions)
Biacore t200 biaevaluation software, by GE Healthcare (1 mentions)
36 protocols using t200 system
1
Thrombin-Gallic Acid Binding Kinetics
2
Measuring CD22 Binding Affinity
3
Kinetic Analysis of Collagen I Binding
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The whole flow path was first primed by running buffer (PBS with 150 mM NaCl, 0.05% surfactant P20, and pH 7.5) for three times. In SPR measurements, reaction temperature was set at 25 °C. Collagen I protein (0.5 mg/ml) in 10 mM sodium acetate buffer (pH 4.5) was immobilized on a carboxymethylated 5 sensor chip. Gradient concentrations of EPS11 (0–15 μM) were injected to Biacore T200 system at a flow rate of 30 μl/min. Results were analyzed with Biacore evaluation software (GE, T200, version 1.0) and fitted to kinetic model. Equilibrium dissociation constant was derived by fitting to a 1:1 Langmuir binding model.
4
Heparan Sulfate Interactions with Heparanase
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The interaction of Hpse with heparin and hs-HA was examined using a BIAcore T200 system. The immobilization of biotinylated GAGs was confirmed by the observation of a ~28 resonance unit increase on a streptavidin-immobilized sensor chip (BR-1005-31, Cytiva, Uppsala, Sweden). The binding reactions were carried out at 20 °C. MuHpse(mature) dissolved in running buffer (HBS-EP+: 10 mM HEPES, 0.15 M NaCl, 3 mM EDTA, and 0.05% (w/v) P20, pH 7.3) was injected onto the sensor chips at a flow rate of 30 µL/min. Association and dissociation times were 120 s and 180 s, respectively. Kinetic parameters were evaluated with BIAevalution software 4.1 (Cytiva, Uppsala, Sweden) using a 1:1 binding model. Association and dissociation rate constants (ka and kd) as well as dissociation equilibrium constants (KD) were determined.
5
Spike Protein Binding Affinity Assay
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The CM5 chip was immobilized with anti-His antibodies using the His Capture Kit (Cytiva) to capture the spike protein through their C-terminal His-tag. Serially diluted human ACE2-Fc protein was then flowed over the chip in HBS-EP + buffer (Cytiva). Binding affinities were measured with the Biacore T200 system at 25°C in the single-cycle mode. Data was analyzed by the Evaluation Software using the 1:1 binding model.
6
Measuring EGFR-MX Binding Affinity
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Surface plasmon resonance binding experiment was performed using Biacore T200 system to analyse the binding affinity between ECD and MX. Human EGFR ECD protein was purchased from Sino Biological (China), and the series s sensor chip CM5 were purchased from Bio. Co. (USA). The ECD protein was immobilized on a CM5 sensor chip and the binding of MX to ECD was accomplished by injecting concentration of MX (250–2000 μM) into the sensor chip under the buffer condition of PBS-buffered saline at 25 °C.
7
Binding of RmPAP to Bovine Hemoglobin
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Refolded RmPAPWT and RmPAPMUT (2.0 µg) were incubated with bovine hemoglobin (5 µg) in 50 mM phosphate-citrate buffer (pH 2.5–6.0) for 4 h at 37 °C and analyzed using SDS-PAGE (15%). The binding of RmPAPWT to bovine hemoglobin was measured by surface plasmon resonance (SPR) using a Biacore T-200 system. Bovine hemoglobin (2000 RFU) was immobilized on a CM5 series chip (FC 2) in acetate buffer (pH 5.5), while BSA was immobilized in FC 1. SPR experiments were conducted by injecting increasing concentrations of RmPAPWT (10 mM phosphate-citrate buffer, pH 4, with 0.15 M NaCl) at 20 µL/min, with association and dissociation times of 300 sec and 900 sec, respectively. The equilibrium constant was determined by plotting the intensity of the steady-state response (FC2 – FC1) against the RmPAP concentration using the Biacore T200 evaluation software (GE Healthcare).
8
Biosensor Analysis of TBK1-Ginsenoside Rd Binding
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The affinity between TBK1 and ginsenoside Rd was analyzed using the Biacore T200 system. Soluble proteins were used as immobilized ligands diluted in 10 mM sodium acetate buffer with pH 4.0. TBK1 protein (OriGene, Maryland, USA) was immobilized on the sensor chip (CM5, GE Healthcare) using the amine-coupling method according to standard protocols. The amount of ligand immobilization was approximately 11000 RU. A similar procedure was performed with reference, but without ligand injection. Subsequently, 1.05 × PBS-P with 5% DMSO solution was used as the running buffer, and ginsenoside Rd with different concentrations was used as the analyte. For binding studies, analytes were applied at corresponding concentrations in flow buffer at a flow rate of 30 μL/min with a contact time of 60 s and a dissociation time of 120 s. The chip platform was washed with flow buffer and 50% dimethyl sulfoxide after each analysis. Biacore T200 evaluation software was performed to analyze the data through curve fitting of a 1:1 binding model.
9
Affinity Characterization of Anti-TNF-α Antibody
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The sample compartment of the Biacore T200 system was set to 20 °C, the analysis temperature to 25 °C, and the data collection rate to 1 Hz. PBS-P+ was used as the running buffer. In each cycle, biotin capture reagent was injected for 300 s at a flow rate of 2 µL/min, followed by a 30–60 s capture of biotinylated TNF-α at 1–2 µg/mL in PBS-P+ with 0.5% BSA, to reach minimum capture levels of around 40 RU. Anti-TNF-α antibody, 0.02–360 µg/mL in PBS-P+, was injected for 120 s and the surface was regenerated at the oligonucleotide level per kit instructions. To study antibody binding to both the captured antigen and a receptor in the same assay, an additional sample injection of receptor was included. Receptors, FcγRIIIa Val 158, FcγRI and TNF-α receptor, were injected for 60 s at concentrations of 5 µg/mL, 5.4 µg/mL and 10 µg/mL, respectively.
Heat stressed antibody samples were analyzed after exposing the antibody at 1 mg/mL to 60 °C for 1, 2 or 3 h prior to analysis.
Data analysis was performed using Microsoft Excel as described insection 2.4 and with the sensorgram comparison functionality in Biacore T200 evaluation software version 3.1.
Heat stressed antibody samples were analyzed after exposing the antibody at 1 mg/mL to 60 °C for 1, 2 or 3 h prior to analysis.
Data analysis was performed using Microsoft Excel as described in
10
Protein Binding Kinetics Characterization
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All experiments were performed in PBS buffer pH 7.4 (NaCl 137 mM, KCl 2.7 mM, Na2HPO4 10 mM, KH2PO4 2 mM, 5% DMSO, and 0.05% Tween 20) on a Biacore T200 System at a flow rate of 30 μL min−1 and 25 °C. YtkR7, Rob, and SbmC proteins were immobilized on a CM5 sensor chip at 23,757.5, 14,397.3, and 3827.0 response units (RU), respectively.
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