SPR analysis of affinity between mouse FcRn and trimeric HA-Fc/wt was performed by the ACROBiosystems (Newark, Delaware, USA). In brief, the purified recombinant FcRn was diluted to 1 μg/mL with 10 mM sodium acetate (pH 4.5) and was immobilized onto a CM5 biosensor chip (Biacore, Uppsala, Sweden) using an amine coupling kit (Biacore). The activator is prepared by mixing 400 mM 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and 100 mM N-Hydroxysuccinimide (NHS) (GE) immediately prior to injection. The CM5 sensor chip is activated for 420 s with the mixture at a flow rate of 10 μL/min, which typically results in immobilization levels of 100 resonance units (RU). The chip is deactivated by 1 M ethanolamine hydrochloride-NaOH (GE) at a flow rate of 10 μL/min for 420 s. The reference surface channel was prepared in the same way as the active surface, but without injecting mouse FcRn. The HA-Fc/wt proteins were diluted with the running buffer B (0.05% Tween-20 in PBS, pH 6.0) to 62.5, 31.25, 15.625, 7.813, 3.906, 1.953 and 0 nM. The HA-Fc/wt proteins were injected and allowed to flow at a rate of 30 μL/min for an association phase of 90 s, followed by 210 s for dissociation in the running buffer B. The affinity was analyzed by Biacore T200 Evaluation Software 3.0 in Biacore T200.
T200 evaluation software 3
Manufactured by Cytiva
The T200 Evaluation Software 3.1 is a software application designed for data analysis and visualization of results obtained from experiments conducted using the T200 Surface Plasmon Resonance (SPR) instrument. The software provides tools for processing, analyzing, and interpreting SPR data.
Automatically generated - may contain errors
Lab products found in correlation
Biacore t200, by Cytiva (13 mentions)
T200 instrument, by Cytiva (2 mentions)
Edc nhs, by GE Healthcare (2 mentions)
Biacore t200, by GE Healthcare (2 mentions)
Amine coupling kit, by Cytiva (1 mentions)
Hbs ep running buffer, by GE Healthcare (1 mentions)
Series s cm5 chip, by Cytiva (1 mentions)
Biacore t100, by Cytiva (1 mentions)
Sensor chip sa, by GE Healthcare (1 mentions)
T200 evaluation software v3, by Cytiva (1 mentions)
Protein a sensor chip, by GE Healthcare (1 mentions)
Screenworks 4, by Molecular Devices (1 mentions)
15 protocols using t200 evaluation software 3
1
FcRn-HA-Fc/wt Affinity Analysis
2
Kinetic Characterization of PfCyRPA Binding
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Experiments were performed at 25°C in the HBS-EP+ running buffer (GE Healthcare). On a series S CM5 chip (Cytiva), an equal amount of rabbit anti-mouse IgG (Cytiva) was immobilized in all four flow cells using standard NHS/EDC chemistry. In the kinetic experiment, 100-150 RU of mAb was captured in Fc 2, Fc 3 and Fc 4, using a flow-rate of 30 µl/min for 30 sec. Next, six different concentrations of PfCyRPA (ranging from 30 nM – 0.12 nM) were injected in duplicate for 300 sec at 60 µl/min in all flow cells. The PfCyRPA analyte was >95% pure as assessed by SDS-PAGE and western blotting. A dissociation time was measured for 600 sec (3600 sec when necessary). Regeneration of the chip was performed using 10 mM glycine-HCl pH 1.7 for 25 sec. Specific binding of PfCyRPA to the captured mAb was obtained by subtracting both the reference flow cell (Fc 1) and a blank run with running buffer only. Sensorgrams were fitted to a global Langmuir 1:1 model, to determine the kinetic association and dissociation rate constants using the Biacore T200 Evaluation Software 3.0. Graphed data show the fold-change in affinity of each mAb to the reference protein 3D7 PfCyRPA relative to the affinity of each mAb to the PfCyRPA variant R339S. Fold-change lower than 1 were plotted as their inverse to allow more uniform data representation compared to fold-change higher than 1.
3
Surface Plasmon Resonance Analysis of Bacterial Antibiotics
4
Surface Plasmon Resonance Binding Affinity of IL-27 and p28
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Surface plasmon resonance experiments were performed to determine the binding affinity of the recombinantly produced IL‐27 and p28 to IL‐27Rα and GP130. These were carried out on a Biacore T100 instrument (T200 sensitivity enhanced). C‐terminal biotinylated mouse GP130 and mouse IL‐27Rα were immobilized onto a SA sensor chip (GE Healthcare) at levels of ~100 response units (RU). The immobilization was performed in 10 mM HEPES, 150 mM NaCl, 0.02% (v/v) TWEEN‐20, pH 7.2 buffer. Analysis runs were performed at 25°C in 10 mM HEPES, 150 mM NaCl, 0.05% (v/v) TWEEN‐20, pH 7.2, and 0.5% BSA. Mouse IL‐27 binding was tested by injecting a 1:3 dilution series of inhibitor from a top concentration of 100 nM, and at a flow rate of 30 μl/min. Mouse p28 binding was tested in a similar manner, but from a top concentration of 20 μM and with a 1:2 dilution series. In both cases, the association time was set to 120 s, while the dissociation time was 600 s for IL‐27 and 60 s for p28. Data analysis was performed using Biacore T200 Evaluation Software 3.0.
5
Kinetic analysis of MERS-PLpro inhibitors
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The MERS-PLpro enzyme was diluted to 50 µg/mL with 10 mM sodium acetate (pH 5.5) and immobilized on a CM5 sensor chip by standard amine-coupling with running buffer PBSP (10 mM phosphate, pH 7.4, 2.7 mM KCl, 137 mM NaCl, 0.05% surfactant P-20) using a Biacore T200 instrument. MERS-PLpro enzyme was immobilized to flow cells 2 and 4, and immobilization levels were ∼10,000 RU for both flow cells. Unmodified flow cells 1 and 3 were used as controls. Compound solutions with a series of increasing concentrations (0–50 µM at 2-fold dilution) were applied to all four channels in SPR binding buffer (PBSP + 0.5 mM TCEP and 2% DMSO) at a 30 µL/min flow rate at 25 °C. Data were double-referenced with both reference cell RU values and zero concentration (2% DMSO) signals, and sensorgrams were analyzed using the Biacore T200 evaluation software 3.0. Response units at each concentration were measured during the equilibration phase for steady-state affinity fittings, and the KD values were determined by fitting the data to a single rectangular hyperbolic curve Eq. (2) , where y is the response, ymax is the maximum response and x is the compound concentration.
Kinetic rate constants were determined by fitting globally to the 1:1 Langmuir model embedded in the Biacore T200 evaluation software v3.0.
Kinetic rate constants were determined by fitting globally to the 1:1 Langmuir model embedded in the Biacore T200 evaluation software v3.0.
6
Quantifying MERS-CoV Protease Binding Kinetics
7
Kinetic Binding Analysis of Anti-Spike mAbs
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For kinetic binding measurements, the CM5 chip surface was activated by injecting a solution of EDC/NHS (GE Healthcare). Mouse anti-human IgG (Fc) mAb (25 μg/ml) was immobilized on the sensor chip by amine coupling, followed by deactivation using 1 M ethanolamine. Afterward, anti-spike mAbs (0.1 μg/ml) were then flowed over and captured on anti-human IgG (Fc) mAb-coated surface. Subsequently, gradient diluted his-tagged SARS-CoV-2 RBD solutions (1.875–30 nM, twofold serial dilution) were injected individually in a single-cycle kinetic format without regeneration (30 μl/min, association:180 s, dissociation:60 s). The binding data were double referenced by blank cycle and reference flow cell subtraction. Processed data were fitted by a 1:1 interaction model using Biacore T200 Evaluation Software 3.1.
8
Kinetic Characterization of SARS-CoV-2 Spike Protein Binding
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Kinetic interactions between the antibodies and his-tagged antigen proteins were measured at room temperature using Biacore T200 surface plasmon resonance (GE Healthcare). Anti-human fragment crystallizable region (Fc region) antibody was immobilized on a CM5 sensor chip to approximately 8,000 resonance units (RU) using standard N‑hydroxysuccinimide/N‑Ethyl-N′-(3-dimethylaminopropyl) carbodiimide hydrochloride (NHS/EDC) coupling methodology. The antibody (1.5 μg/mL) was captured for 60 s at a flow rate of 10 μL/minute. The SARS-CoV-2 Spike S1, SARS-CoV-2 (2019-nCoV) Spike S1- B.1.1.7 lineage mut (HV69-70 deletion, Y144 deletion, N501Y, A570D, D614G, P681H)-His and SARS-CoV-2 (2019-nCoV) Spike S1- B.1.351 lineage mut (K417N, E484K, N501Y, D614G)-His proteins were run at six different dilutions in a running buffer of 0.01 M HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.05% v/v Surfactant P20 (HBS‑EP +). All measurements were conducted in HBS-EP + buffer with a flow rate of 30 μL/min. The affinity of antibody was analyzed with Biacore T200 Evaluation software 3.1. A 1:1 (Langmuir) binding model was used to fit the data.
9
Kinetic Binding of Anti-Spike mAbs
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For kinetic binding measurements, CM5 chip surface was activated by injecting a solution of EDC/NHS (GE Healthcare). Mouse anti-human IgG (Fc) mAb (25 μg/ml) was immobilized on the sensor chip by amine coupling, followed by deactivation using 1M ethanolamine. Afterward, anti-spike mAbs (0.1 μg/ml) were then flowed over and captured on anti-human IgG (Fc) mAb-coated surface. Subsequently, gradient diluted his-tagged SARS-CoV-2 RBD solutions (1.875 nM-30 nM, two-fold serial dilution) were injected individually in single-cycle kinetic format without regeneration (30 μl/min, association:180s, dissociation:60s). The binding data were double referenced by blank cycle and reference flow cell subtraction. Processed data were fitted by 1:1 interaction model using Biacore T200 Evaluation Software 3.1.
10
Quantifying Antibody-OX40 Antigen Affinity
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Surface plasmon resonance (SPR) analysis was carried out using Protein A sensor chips (GE Healthcare) for measuring affinity kinetics between antibodies and OX40 antigen. Antibodies were diluted with HBS EP+ running buffer to 5 µg/ml and were first immobilized onto the sample flow cell of Protein A sensor chip with a flow rate of 10 μl/min at 25°C, and the reference flow cell was left blank. OX40 antigens were serially diluted with HBS EP+ running buffer, then injected over the two flow cells at a range of eight concentrations using a single-cycle kinetics program. HBS EP+ running buffer was also injected using the same program for background subtraction. All data were fitted to a 1:1 binding model using Biacore T200 Evaluation Software 3.1.
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