All experiments were done at 25°C using a Biacore T200, equilibrated in running buffer containing 25 mM Tris–HCl (pH 7.6), 300 mM NaCl, 0.5 mM TCEP, and 0.005% (vol/vol) Tween-20. CM-5 chips with anti-GST-nanobodies (GST VHH; Chromotech) were used to couple GST-MDM2 variants to a level of ∼500 response units, with GST as a control. UBE2D2S22R C85K–Ub (UBE2D2–Ub), used as an analyte, was purified as described previously (Buetow et al, 2015 (link)) and serially diluted in running buffer. Binding between GST-MDM2 variants and UBE2D2–Ub was measured in duplicate, across seven concentrations of UBE2D2–Ub, starting at 100 μM and decreasing in a twofold manner. Data shown are the difference between SPR signal recorded for GST-MDM2 variants and GST alone. The data were analyzed by steady-state affinity using Biacore T200 evaluation software (GE Healthcare) and plotted in Prism8 (GraphPad).
Biacore t200 evaluation software
Manufactured by GE Healthcare
Sourced in United States, Sweden
The Biacore T200 evaluation software is a tool used to analyze and interpret data generated by the Biacore T200 system, a surface plasmon resonance (SPR) instrument. The software enables users to perform real-time interaction analysis, kinetic studies, and affinity measurements of biomolecular interactions.
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Lab products found in correlation
Biacore t200, by GE Healthcare (39 mentions)
Cm5 sensor chip, by GE Healthcare (11 mentions)
Biacore t200 system, by GE Healthcare (10 mentions)
Biacore t200 instrument, by GE Healthcare (7 mentions)
Amine coupling kit, by GE Healthcare (5 mentions)
Biacore t100 sensitivity enhanced t200 equipment, by GE Healthcare (4 mentions)
Biacore t200, by Cytiva (3 mentions)
Cm5 chip, by GE Healthcare (3 mentions)
Biacore t200 spr instrument, by GE Healthcare (3 mentions)
Biacore t200 spr system, by GE Healthcare (3 mentions)
Series s sensor chip cm5, by GE Healthcare (3 mentions)
Prism 8, by GraphPad (3 mentions)
Hbs ep, by GE Healthcare (2 mentions)
Biacore t200 spr system, by Cytiva (2 mentions)
Hbs ep buffer, by GE Healthcare (2 mentions)
Biacore 2000, by GE Healthcare (2 mentions)
Biacore t200 optical biosensor, by GE Healthcare (2 mentions)
Glycine hcl, by GE Healthcare (2 mentions)
Series s sensor chip sa, by GE Healthcare (2 mentions)
Glycine 1, by GE Healthcare (2 mentions)
90 protocols using biacore t200 evaluation software
1
MDM2 Interaction Kinetics with UBE2D2-Ub
2
Quantifying VDR-RXR Interactions by SPR
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Measurements were performed by Biacore T100 sensitivity enhanced T200 equipment (GE Healthcare) using CM5 series S sensor chip (GE) (29-1496-03). MED1(50–660) was immobilized on the chip surface using standard amino-coupling protocol in 10 mM Na-acetate buffer pH 5.5. The resulting immobilized MED1 was in the range of 100–200 response unit. The running buffer was 50 mM HEPES pH 7.5, 400 mM NaCl, 1 mM TCEP, 0.005% Tween 20 and for regeneration 1 M sodium chloride solution was used. Interactions of the MED1 with fully liganded VDR–RXR wild type, VDRΔH12–RXR, VDR–RXR AF-2 mutant and VDR–RXR ΔH12 were analyzed in the manner of dose response using twofold dilution series of VDR–RXR ranging from 0.01 to 8 μM. The association phase was 120 s and the dissociation phase was 120 s. After subtracting the reference and buffer signal, the data were fit to a steady state binding model using the Biacore T200 Evaluation software (GE Healthcare).
3
Quantifying 5D12-hCLDN-4 Binding Kinetics
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To determine the binding kinetics of 5D12 to hCLDN‐4, surface plasmon resonance analysis (Biacore T200, GE Healthcare, Little Chalfont, UK) was performed as described previously (Li et al. 2014a ). Briefly, anti‐rat IgG (BD Biosciences) was immobilized on a CM5 sensor chip to approximately 5000 resonance units by using N‐(3‐dimethylaminopropyl)‐N′‐ethylcarbodiimide and N‐hydroxysuccinimide as activating reagents; remaining reactive groups were blocked with ethanolamine. The 5D12 antibody was immobilized on a CM5 sensor chip by using anti‐rat IgG. The subsequent binding experiment used His‐tagged hCLDN‐4 (purified by using an Sf‐9 cell expression system) as an analyte at concentrations ranging from 10 to 500 nmol/L (Uchida et al. 2010 ). Within a single binding cycle, the analyte was injected sequentially in order of increasing concentration over both the ligand and the reference surfaces. The reference surface, an unmodified flow cell, was used to correct for systematic noise and instrumental drift. The sensorgrams were globally fitted by using a 1:1 binding model to determine the ka, kd, and KD values (Biacore T200 Evaluation Software, GE Healthcare).
4
Kinetics and Affinity of AM2H10 Binding
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The binding kinetics and affinity of AM2H10 to M2-nanodiscs, empty nanodiscs, and synthesized M2e (ectodomain of M2) were measured by SPR on the Biacore T200 instrument (GE Healthcare). Subsequently, 10 μg/mL of AM2H10 was captured onto a CM5 sensor chip in 10 mM sodium acetate buffer at pH 4.0 for 200 sat 10 μL/min. At the end of each cycle, the running buffer flowed for 300 s, followed by 50 mM NaOH for regenerating the surface of the CM5 chip. Biacore data were analyzed using the Biacore T200 Evaluation Software (GE Healthcare Bio-Sciences AB) according to the method described previously.77 (link) The 1:1 Langmuir binding model with mass transfer control was used and data were fed into a global fit to extract the binding characteristics.
5
CK1α Interaction Kinetics by SPR
6
Quantifying Influenza Vaccine Antigenicity
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Antigenicity of the influenza formulations was quantified by a SPR method modified from Estmer Nilsson et al[15] (link). Samples were analyzed on a Sensor Chip CM5 with a Biacore T200 biosensor system (GE Healthcare). HBS-EP+ (GE Healthcare) was used as analysis buffer. Recombinant HA protein from A/PR/8/34 (Protein Sciences) was immobilized to 7000–10000 response units using an Amine coupling kit (GE Healthcare) with ∼65 μL rHA (10 μg/mL) in 10 mM phosphate buffer, 0.05% Surfactant P20 (GE Healthcare), pH 6.0. Dilutions series of the vaccine samples were made, and anti-influenza A/PR/8/34 sheep serum (1∶150, NIBSC) was added to each dilution. The sample-serum mixture was subsequently injected during 400 seconds during which sensorgrams were acquired. In between each sample the sensor chip surface was regenerated using 50 mM HCl, 0.05% Surfactant P20. Acquired sensorgrams were analyzed using Biacore T200 evaluation software (GE Healthcare). Antigenicity was calculated relative to a known concentration of rHA A/PR/8/34.
7
Sesquiterpene-Hemoglobin Interaction Analysis
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The interaction of representative sesquiterpenes in ZTOI between Hb was detected using SPR (Biacore T200, GE Healthcare). Hb was dissolved in HBS‐N buffer (0.01 M HEPES, pH 7.4, 0.15 M NaCl, GE Healthcare) and diluted to 100 μg/ml with 10 mM acetate (pH 5.5). The amine coupling kit (GE Healthcare) was used to immobilize Hb on a CM5 sensor chip (Series S, Cytiva). According to recommendation of manufacturer, 1‐ethyl‐3‐(3‐dimethylaminopropyl)‐carbodiimide hydrochloride (EDC) and N‐hydroxysuccinimide (NHS) were used to activate the chip, and residual activated groups were blocked by 1.0 M ethanolamine‐HCl (pH 8.5). The running buffer was 1.05× PBS‐P containing 5% DMSO (v/v) and 0.02% surfactant P20 (v/v). Each sesquiterpene was accurately weighed and dissolved in DMSO to prepare stock solutions. The sensorgrams were fitted by the 1:1 binding model to calculate equilibrium dissociation constant (KD) in Biacore T200 Evaluation Software (version 3.2, General Electric Company).
8
Kinetic Analysis of ANGPTL3-FLD Antibody
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Surface plasmon resonance (SPR) experiments were performed using a Biacore T200 machine at 25 °C. HBS-EP was used as the running buffer. In these measurements, the ANGPTL3-FLD recombinant protein was coupled on a CM5 chip at pH 5.0 using an amine coupling kit (GE Healthcare, USA). Samples at a 6.25–800 nM concentration were captured on the second flow cell at a flow rate of 30 μl/min. A dilution series of the 5E5F6 monoclonal antibody was passed through both flow cells at 30 μl/min to record the association phase (180 s). The dissociation phase was monitored for 480 s and triggered by replacing the sample solution with HBS-EP. After each cycle, the sensor surface was regenerated with a short treatment using ten mM glycine-HCl (pH 2.1). Biacore T200 evaluation software (GE Healthcare, USA) was used to record and analyze the binding kinetics using the 1:1 binding model.
9
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).
10
Kinetic Analysis of C1r Binding to FbpC-C
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Surface plasmon resonance (SPR) was performed on a Biacore T200 instrument at 25°C using a flowrate of 30 μL/min. HBS-T running buffer was used (10 mM HEPES (pH 7.3), 140 mM NaCl, and 0.005% Tween 20) supplemented with 5 mM CaCl2. FbpC-C was immobilized on a CMD200 sensor chip (Xantec Bioanalytics) via standard amine coupling as before (20 (link), 30 (link)–32 (link)). The immobilization density for FbpC-C was 1,138.2 RU, FbpC-C R248A was 975.2 RU, and FbpC-C H252A was 1,082 RU. Zymogen or active C1r binding data was obtained using a single cycle injection strategy (20 (link)) whereby a fivefold dilution series of either C1r zymogen or active C1r ranging from 0–100 nM was injected over the immobilized FbpC-C biosensor. Data was obtained in triplicate with each cycle having association times of 5 min, and a final dissociation time of 1 hour. Surfaces were regenerated to baseline with two 1 min injections with regeneration buffer (0.1 M Glycine [pH 2.2], 2.5 M NaCl). Equilibrium dissociation constants (KD) and kinetic rates were obtained from the resulting kinetic fits obtained using BiacoreT200 Evaluation Software (GE Healthcare).
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