The refolded hACE2, eqACE2 and mutated eqACE2 were transferred into PBST buffer (1.8 mM KH2PO4, 10 mM Na2HPO4 (pH 7.4), 137 mM NaCl, 2.7 mM KCl and 0.05% (v/v) Tween 20) and biotinylated with an NHS-LC-LC-Biotin kit. Then, the biotinylated proteins are immobilized on flow cell 2 of a SA chip. Flow cell 1 was used as the negative control. Then, serially diluted wt or mutated RaTG13-RBD and SARS-COV-2 PT-RBD were flowed over the chip in PBST buffer. Binding affinities were measured using a BIAcore 8 K (GE Healthcare) at 25 °C in the single-cycle mode. Binding kinetics data were collected using BIAcoreTM 8 K control software 3.0.12.15655 and analyzed with BiacoreTM Insight software (GE healthcare) using a 1:1 Langmuir binding model. The SARS-COV-2 PT-RBD, RBDs from VOCs and mutated SARS-COV-2 PT-RBD were immobilized on CM5 chips and the following protocol of SPR assay is the same.
Biacore 8k
Manufactured by GE Healthcare
Sourced in United States, Sweden, United Kingdom
The Biacore 8K is a label-free biomolecular interaction analysis system from GE Healthcare. It is designed to measure real-time interactions between molecules, providing insights into binding kinetics, affinity, and specificity. The system utilizes surface plasmon resonance (SPR) technology to detect and analyze these interactions without the need for labeling.
Automatically generated - may contain errors
Lab products found in correlation
Biacore 8k evaluation software, by GE Healthcare (11 mentions)
Biacore evaluation software, by GE Healthcare (10 mentions)
Cm5 chip, by GE Healthcare (7 mentions)
Cm5 sensor chip, by GE Healthcare (7 mentions)
Biacore insight software, by GE Healthcare (6 mentions)
Series s sensor chip cm5, by GE Healthcare (5 mentions)
Biacore insight evaluation software, by GE Healthcare (5 mentions)
Protein a sensor chip, by GE Healthcare (5 mentions)
Surfactant p20, by GE Healthcare (4 mentions)
Amine coupling kit, by GE Healthcare (4 mentions)
Recombinant protein a, by Sino Biological (3 mentions)
Protein a chip, by GE Healthcare (3 mentions)
Hbs ep buffer, by GE Healthcare (3 mentions)
Series s sensor chip sa, by GE Healthcare (3 mentions)
Series s sensor chip, by GE Healthcare (3 mentions)
Biacore 4000, by GE Healthcare (2 mentions)
Biacore t200, by GE Healthcare (2 mentions)
Biaevaluation 4, by GE Healthcare (2 mentions)
Hbs ep running buffer, by GE Healthcare (2 mentions)
Ez link maleimide peg2 biotin, by Thermo Fisher Scientific (2 mentions)
133 protocols using biacore 8k
1
Characterizing SARS-CoV-2 RBD Binding Affinities
2
SARS-CoV-2 RBD Binding Affinity Assay
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We tested the binding affinities between the mFc-tagged ACE2s and SARS-CoV-2 RBD or SARS-CoV RBD proteins by SPR using a BIAcore 8K (GE Healthcare) at 25°C in single-cycle mode. SARS-CoV-2 NTD protein was used as a negative control. The HBS-EP buffer (20 mM HEPES, pH 7.4, 150 mM NaCl and 0.005% (v/v) Tween 20) was used as the running buffer, and SARS-CoV-2 RBD, SARS-CoV RBD and SARS-CoV-2 NTD proteins were exchanged into this buffer by gel filtration before use. First, the anti-mFc antibodies were immobilized on the CM5 biosensor chip (GE Healthcare) using amine-coupling chemistry (GE Healthcare). Then, the supernatants containing mFc-tagged ACE2s were injected and captured respectively at ∼100–700 response units. SARS-CoV-2 RBD, SARS-CoV RBD or SARS-CoV-2 NTD protein was serially diluted and flowed through the chip surface and the binding response was measured. The anti-mFc antibody was regenerated with 10 mM Glycine-HCl (pH 1.7). The equilibrium dissociation constants (KD) of each interaction pair were calculated using BIAcore 8K Evaluation Software (GE Healthcare) by fitting to a 1 : 1 Langmuir binding model. Supernatant containing hACE2-mFc protein was used as a positive control.
3
Characterizing ACE2-RBD Binding Kinetics
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The 25 ACE2-mFC fusion proteins were transferred into HBST buffer (20 mM HEPES (pH 7.4), 150 mM NaCl, and 0.005% (v/v) Tween 20) and immobilized on the CM 5 chip (Table S2 ). Then, serially diluted wild-type or mutated RaTG13 RBD proteins flowed over the chip in HBST buffer. PD-L1 protein was used as negative control. Binding affinities were measured using a BIAcore 8K (GE Healthcare) at 25°C in the single-cycle mode. Binding kinetics were analyzed with Biacore™ Insight software (GE healthcare) using a 1:1 Langmuir binding model.
The seven recombinant antibodies (2 μg/mL) were first captured on flow cell 2 of the protein A sensor chip (GE Healthcare) at more than 500 response units (Table S4 ). Flow cell 1 was used as the negative control. Then, serially diluted SARS-CoV-2 RBD or RaTG13 RBD proteins flowed over the chip in PBST buffer. Response Units (RU) were measured with a BIAcore 8K (GE Healthcare) at 25°C in single-cycle mode. The antibodies were regenerated with 10 mM glycine-HCl (pH 1.5). The equilibrium dissociation constants (KD) of each pair of interactions were calculated using BIAcore® 8K Evaluation Software (GE Healthcare) by fitting to a 1:1 Langmuir binding model.
The seven recombinant antibodies (2 μg/mL) were first captured on flow cell 2 of the protein A sensor chip (GE Healthcare) at more than 500 response units (
4
Biacore 8K Antibody Affinity Assay
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A Biacore 8K instrument (GE Healthcare, Glattbrugg, Switzerland) was used to determine the apparent dissociation constant (KD) of antigen-specific human polyclonal antibodies to P27A. Around 10 μg/mL of P27A in 10 mM sodium acetate, pH = 4.5 was immobilized on a CM5 chip to reach the final chip density of about 150 response units (RU). Affinity-purified antibodies from volunteers were 1:2 serially diluted from 1 μM down to 200 pM and injected at a flow rate of 10 μL/min. After each cycle of injection, the surface chip was regenerated by flushing it with 3 M MgCl for two consecutive 30-second intervals. Association (kon), dissociation (koff) and equilibrium (KD) constants were determined using 1:1 Langmuir binding kinetic fits provided by the suite of analysis software from Biacore 8K (GE Healthcare #29310604).
5
Quantifying Nanobody-RBD Interactions
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The affinity of anti-RBD nanobodies and RBD antigen was measured with Biacore 8 K. A biosensor chip, Series S Sensor Chip Protein A (Cat. # 29127556, GE), was used to affinity capture a certain amount of Fc-tagged nanobodies to be tested and then flow through a series of COVID-19 S.P. RBD (Cat. # 40592-V08B, SinoBiological) under a concentration gradient on the surface of the chip (dilution ratio: 2, conc. levels: at least 5 (excluding curves with irregularities or high background)). Biacore 8 K (GE) was used to detect the reaction signal in real-time to obtain the binding and dissociation curves.
To measure the competitive response of anti-RBD nanobodies and hACE2, Fc-tagged nanobodies were immobilized on chip, then flowed with a 50 nM of RBD (Cat. # 40592-V08B, SinoBiological) and a 100 nM of hACE2 (Cat. #1010B-H08H, SinoBiological). the reaction signal in real-time were detected to obtain the binding and dissociation curves (theoretical ACE2 Rmax > 220 RU and kinetically simulated ACE2 binding > 160 RU for all). The buffer used in the experiment is an HBS-EP + solution (pH 7.4, Cat. # BR100669, GE). The data obtained in the experiment was fitted with Biacore Insight Evaluation Software v3.0, GE software with a (1:1) binding model to obtain the affinity value.
To measure the competitive response of anti-RBD nanobodies and hACE2, Fc-tagged nanobodies were immobilized on chip, then flowed with a 50 nM of RBD (Cat. # 40592-V08B, SinoBiological) and a 100 nM of hACE2 (Cat. #1010B-H08H, SinoBiological). the reaction signal in real-time were detected to obtain the binding and dissociation curves (theoretical ACE2 Rmax > 220 RU and kinetically simulated ACE2 binding > 160 RU for all). The buffer used in the experiment is an HBS-EP + solution (pH 7.4, Cat. # BR100669, GE). The data obtained in the experiment was fitted with Biacore Insight Evaluation Software v3.0, GE software with a (1:1) binding model to obtain the affinity value.
6
SARS-CoV-2 Variant RBD Binding Kinetics
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The SPR assays were performed to test the interactions between mFc‐fused human ACE2 and SARS‐CoV‐2 variant RBDs using a BIAcore 8 K (GE Healthcare) with a CM5 chip (GE Healthcare) at 25°C in single‐cycle mode. SARS‐CoV‐2 WT RBD was used as a positive control. All proteins used for kinetic analysis were exchanged to the PBST buffer (10 mM Na2HPO4, 2 mM KH2PO4, pH 7.4, 137 mM NaCl, 2.7 mM KCl, 0.005% Tween 20) and the anti‐mIgG antibody (Cytiva) was preimmobilized on the CM5 chip using standard amine coupling chemistry with a 50 μg/ml concentration. Concentrated supernatant containing hACE2‐mFc protein was captured on the chip using this immobilized antibody. Various concentrations of RBDs (6.25, 12.5, 25, 50, 100 nM) then flowed through the chip and the real‐time response was recorded to evaluate hACE2 binding. The chip was regenerated after each reaction using glycine (pH 1.7). The equilibrium dissociation constants (binding affinity, KD) for each pair of interactions were calculated using BIAcore 8K® evaluation software (GE Healthcare). The KD values were calculated using the model of 1:1 (Langmuir) binding mode. These results were then visualized using Origin 2021.
7
Fab-Protein Binding Kinetics via SPR
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SPR measurements were performed on a Biacore 8K (GE Healthcare) with HBS-EP+ as running buffer (10 mM HEPES pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.005% v/v Surfactant P20, GE Healthcare) at room temperature. Approximately 700 response units (RU) of Fabs were immobilized on a CM5 sensor chip (GE Healthcare) via amine coupling, and designed monomeric proteins were injected as analyte in two-fold serial dilutions. The flow rate was 30 μl/min with 120 s of contact time followed by 400 s dissociation time. After each injection, surface was regenerated using 0.1 M glycine at pH 3.5. Data were fitted using 1:1 Langmuir binding model within the Biacore 8K analysis software (GE Healthcare #29310604).
8
SPR-Based Kinetic Analysis of Compound Binding
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Experiments were performed on a BiacoreTM 8k instrument (GE Healthcare). Biotinylated cIAP1Bir3 was captured onto a streptavidin sensor chip to levels ranging from 3000–4000 RU. Compound binding experiments were performed in 10 mM HEPES, pH 7.5, 150 mM NaCl, 0.02% BSA, 0.01% P20, and 2% DMSO at 25 °C. Experiments were carried out with n = 4 at 6 concentrations, with the highest concentration of 37 nM and subsequent 3-fold dilutions. BCCov was injected at a flow rate of 30 µL/min, total contact time of 120 s, and dissociation times of 5000 s, using the Single Cycle Kinetics experiment. Binding responses were processed using the Biacore 8k software to zero, x-align, double reference, and correct for excluded volume effects of DMSO in the data.
Note, as shown in Supplementary Fig.6C , a higher concentration of injectant (12 nM) showed a slower on-rate and overall response unit signal due to incomplete removal of the initial 4 nM injectant. These data suggest even with extensive washes, the slow off-rate kinetics do not permit complete removal of compound, making subsequent analysis difficult.
Note, as shown in Supplementary Fig.
9
FtsZ Interaction with AP-3 Binding
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The interaction and dissociation constants between FtsZ and AP-3 were determined by surface plasmon resonance biosensor analysis using BiacoreTM 8K biosensor (GE Healthcare) at 25 °C. For molecule interaction determination, the purified FtsZ was bound to the Series S sensor chip CM5. In detail, 20 μg/mL FtsZ in 10 mM NaAc (pH 4.0) was coupled to the CM5 chip at 6000 RU, then AP-3 solutions of 0–100 μM were flew through the chip, and signal responses were recorded. The signal of solution without AP-3 was used as a blank control, and the value was subtracted from other values during data processing.
10
Protein-Ligand Binding Kinetics via SPR
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Surface plasmon resonance biosensor analysis system was performed with a Biacore TM 8 K (GE Healthcare) for monitoring the biomolecular interactions at 25 °C. The buffer containing the purified proteins was exchanged with PBS for preventing interference to the detection signal during the test. Then, the proteins were diluted to a concentration of 10 µg/mL with 10 mM sodium acetate buffer (pH 4.0) and then immobilized on the surface of CM5 sensor chips. AP-3 was dissolved in PBS buffer with 0.5% DMSO and diluted to gradient concentrations of 100, 50, 25, 12.5 and 6.25 μM. During the binding process, AP-3 solution flowed across the CM5 surface in PBS buffer at a flow rate of 30 μL · min−1 for 90 s. Then in the dissociation step, PBS buffer was passed across the surface of CM5 at a flow rate of 30 μL · min−1 for 120 s to elute AP-3. For every purified protein, responses signals from experiments with gradient concentrations of AP-3 were recorded, and the affinity constant (KD) was calculated by Biacore Insight Evaluation Software.
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