The binding affinity of RSL3-minus-Cl for GPX4 was assayed using the SPR-based Biacore X100 instrument (Cytiva). His-tagged GPX4U46C was immobilized on a CM5 sensor chip by using His Capture Kit (Cytiva, Cat# 28995056) under standard condition at 25 °C with running buffer HBS-EP+ (10 mM HEPES, pH 7.4, 150 mM NaCl, 3 mM EDTA, and 0.05% v/v surfactant P-20). A reference flow cell was activated and blocked in the absence of GPX4. The GPX4 immobilization level was fixed at 1,000 response units (RU), and then different concentrations of RSL3-minus-Cl were serially injected into the channel to evaluate binding affinity. Between injections of RSL3-minus-Cl samples of different concentrations, regeneration of sensor chip was performed by washing with the regeneration buffer (20 mM Glycine, pH 1.5) provided by the same His Capture Kit (Cytiva, Cat# 28995056). The equilibrium dissociation constant (KD) of the RSL3-minus-Cl was obtained by fitting binding response units to the Hill equation.
His capture kit
Manufactured by Cytiva
Sourced in United States
The His Capture Kit is a laboratory equipment product designed for the purification of histidine-tagged (His-tagged) proteins. It provides a simple and efficient method for capturing and isolating His-tagged proteins from complex samples, such as cell lysates or culture supernatants.
Automatically generated - may contain errors
Lab products found in correlation
Biacore t200, by Cytiva (5 mentions)
Hbs ep buffer, by Cytiva (3 mentions)
Series s cm5 chip, by Cytiva (3 mentions)
Series s sensor chip cm5, by Cytiva (3 mentions)
Biacore 8k, by Cytiva (3 mentions)
Biacore 8k insights evaluation software, by Cytiva (2 mentions)
T200 biosensor, by Cytiva (2 mentions)
Biacore x100, by Cytiva (1 mentions)
Biacore 8k+, by Cytiva (1 mentions)
T200 system, by Cytiva (1 mentions)
Cm5 sensor chip, by Cytiva (1 mentions)
Protein a sensor chip, by Cytiva (1 mentions)
Biacore insight evaluation software, by Cytiva (1 mentions)
Biacore insight, by Cytiva (1 mentions)
Series s sensor chip protein a, by Cytiva (1 mentions)
Biotin capture kit, by Cytiva (1 mentions)
Recombinant proteins, by Sino Biological (1 mentions)
Biacore sensor chips, by Cytiva (1 mentions)
Cm5 chip, by Cytiva (1 mentions)
11 protocols using his capture kit
1
SPR Analysis of RSL3-GPX4 Binding
2
SPR Assay for SARS-CoV-2 IgG Binding
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SPR binding assays for IgG binding to S protein were performed using a Biacore T200 biosensor, equipped with a Series S CM5 chip (Cytiva), in a running buffer of 10 mM HEPES pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.05% P-20 (HBS-EP+ buffer, Cytiva) at 25°C. SARS-CoV-2 or SARS-CoV S protein was captured through its C-terminal His-tag over an anti-His antibody surface. These surfaces were generated using the His-capture kit (Cytiva) according to the manufacturer’s instructions, resulting in approximately 10,000 resonance units (RU) of anti-His antibody over each surface. S protein was captured over a single flow cell at 125 to 200 RU. An anti-His antibody surface was used as a reference flow cell to remove bulk shift changes from the binding signal. IgG antibodies were tested using a three-fold dilution series of IgG antibodies with concentrations ranging from 1.2 nM to 33.3 nM. The association and dissociation rates were each monitored for 55 s and 300 s respectively, at 50 mL/minute. The bound S protein-IgG complexes were regenerated from the anti-His antibody surface using 10 mM glycine pH 1.5. Blank buffer cycles were performed by injecting running buffer instead of IgG to remove systematic noise from the binding signal. The resulting data was processed and fitted to a 1:1 binding model using Biacore Evaluation Software.
3
Serum Epitope Mapping Assay Protocol
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Serum epitope mapping competition assays were performed, as previously described (Corbett et al., 2021a ), using the Biacore 8K+ surface plasmon resonance system (Cytiva). Briefly, through primary amine coupling using a His capture kit (Cytiva), anti-histidine antibody was immobilized on Series S Sensor Chip CM5 (Cytiva) allowing for the capture of his-tagged SARS-CoV-2 S-2P on active sensor surface.
Human IgG monoclonal antibodies (mAbs) used for these analyses include: RBD-specific mAbs B1-182, A19-46.1, A19-61.1, S309, A23-97.1, and A23-80.1. Negative control antibody or competitor mAb was injected over both active and reference surfaces. Following this, NHP sera (diluted 1:50) was flowed over both active and reference sensor surfaces. Active and reference sensor surfaces were regenerated between each analysis cycle.
For analysis, sensorgrams were aligned to Y (Response Units) = 0, using Biacore 8K Insights Evaluation Software (Cytiva) beginning at the serum association phase. Relative “analyte binding late” report points (RU) were collected and used to calculate fractional competition (% C) using the following formula: % C = [1 – (100 ∗ ( (RU in presence of competitor mAb) / (RU in presence of negative control mAb) ))]. Results are reported as fractional competition. Assays were performed in duplicate, with average data point represented on corresponding graphs.
Human IgG monoclonal antibodies (mAbs) used for these analyses include: RBD-specific mAbs B1-182, A19-46.1, A19-61.1, S309, A23-97.1, and A23-80.1. Negative control antibody or competitor mAb was injected over both active and reference surfaces. Following this, NHP sera (diluted 1:50) was flowed over both active and reference sensor surfaces. Active and reference sensor surfaces were regenerated between each analysis cycle.
For analysis, sensorgrams were aligned to Y (Response Units) = 0, using Biacore 8K Insights Evaluation Software (Cytiva) beginning at the serum association phase. Relative “analyte binding late” report points (RU) were collected and used to calculate fractional competition (% C) using the following formula: % C = [1 – (100 ∗ ( (RU in presence of competitor mAb) / (RU in presence of negative control mAb) ))]. Results are reported as fractional competition. Assays were performed in duplicate, with average data point represented on corresponding graphs.
4
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.
5
Quantifying IgG Binding to SARS-CoV-2 Spike
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SPR binding assays for IgGs binding to spike, were performed using a Biacore T200 biosensor, equipped with a Series S CM5 chip (cat# BR100030, Cytiva, MA), in a running buffer of 10mM HEPES pH 7.4, 150mM NaCl, 3 mM EDTA, 0.05% P-20 (HBS-EP+ buffer, Cytiva, MA) at 25°C. SARS-CoV-2 spike protein was captured through its C-terminal his-tag over an anti-his antibody surface. These surfaces were generated using the His-capture kit (Cat#: 28995056, Cytiva, MA) according to the instructions of the manufacturer, resulting in approximately 10,000 RU of anti-his antibody over each surface. Spike protein was captured over a single flow cell at a capture level of 125-200 RU. An anti-his antibody surface was used as a reference flow cell to remove bulk shift changes from the binding signal.
IgGs were tested using a three-fold dilution series of IgGs with concentrations ranging from 33.3 nM to 1.2 nM. The association and dissociation rates were each monitored for 55 s and 300 s respectively, at 50 μL/min. The bound spike/IgG complex was regenerated from the anti-his antibody surface using 10 mM Glycine pH 1.5. Blank buffer cycles were performed by injecting running buffer instead of IgG to remove systematic noise from the binding signal. The resulting data was processed and fit to a 1:1 binding model using Biacore Evaluation Software.
IgGs were tested using a three-fold dilution series of IgGs with concentrations ranging from 33.3 nM to 1.2 nM. The association and dissociation rates were each monitored for 55 s and 300 s respectively, at 50 μL/min. The bound spike/IgG complex was regenerated from the anti-his antibody surface using 10 mM Glycine pH 1.5. Blank buffer cycles were performed by injecting running buffer instead of IgG to remove systematic noise from the binding signal. The resulting data was processed and fit to a 1:1 binding model using Biacore Evaluation Software.
6
SARS-CoV-2 RBD Binding Kinetics
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SPR experiments were performed using Biacore 8K (Cytiva). SA55 or SA58 (human IgG1) was captured by a Sensor Chip Protein A (Cytiva), and SARS-CoV-2 WT or BA.5 RBD (His Tag, Sino Biological) of various concentration (1.5625 nM, 3.125 nM, 6.25 nM, 12.5 nM, 25 nM, and 50 nM) were injected. The response was recorded at room temperature, and the raw data curves were fitted to a 1:1 binding model using Biacore Insight Evaluation Software (Cytiva, v4.0.8).
For competition assays, SARS-CoV-2 WT or BA.5 RBD (His Tag, Sino Biological) was captured by a Sensor Chip CM5 (Cytiva) with immobilized anti-His using His Capture Kit (Cytiva), and SA55 or SA58 (200 nM) was injected. After equilibrium, the other mAb was also added (SA58+SA55, 200 nM each). The response was recorded by Biacore Insight Evaluation Software (Cytiva, v4.0.8) at room temperature. For control, we also determined the responses using SA55 or SA58 (200 nM) only.
For competition assays, SARS-CoV-2 WT or BA.5 RBD (His Tag, Sino Biological) was captured by a Sensor Chip CM5 (Cytiva) with immobilized anti-His using His Capture Kit (Cytiva), and SA55 or SA58 (200 nM) was injected. After equilibrium, the other mAb was also added (SA58+SA55, 200 nM each). The response was recorded by Biacore Insight Evaluation Software (Cytiva, v4.0.8) at room temperature. For control, we also determined the responses using SA55 or SA58 (200 nM) only.
7
SARS-CoV-2 spike protein binding assay
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The following Biacore sensor chips and reagents were purchased from Cytiva (Marlborough, MA, USA): Series S sensor chip CM5, Series S sensor chip protein A, Biotin CAPture Kit containing Sensor Chip CAP, Biotin CAPture Reagent, regeneration solution (8 M guanidine hydrochloride and 1 M sodium hydroxide), His-Capture Kit containing anti-histidine antibody, immobilization buffer (10 mM sodium acetate, pH 4.5), and regeneration solution (10 mM glycine, pH 1.5). SARS-CoV-2 spike S1 rabbit mAb1, SARS-CoV-2 spike neutralizing rabbit mAb2, and recombinant proteins corresponding to the original SARS-CoV-2 spike RBD; original S1; spike S1 variant D614G; and RBD protein variants N501Y, N439K, Y453F, and E84K were purchased from Sino Biological (Wayne, PA, USA). The recombinant human ACE2 receptor was purchased from Acro Biosystems (Cambridge, MA, USA). The specifications of the materials are presented in Table S1 .
8
Serum Antibody Epitope Mapping Assay
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Serum antibody epitope mapping competition assays were performed as previously described.39 (link),83 (link) Briefly, primary amine coupling was used to immobilize anti-histidine antibody on a Series S Sensor Chip CM5 (Cytiva) via His capture kit (Cytiva). His-tagged SARS-CoV-2 S-2P was then captured on the sensor surface. Following this, NHP sera (diluted 1:50) was flowed over both active and reference sensor surfaces. Active and reference sensor surfaces were regenerated between each analysis cycle. Human IgG monoclonal antibodies (mAb) used for these analyses include: S2-specific mAbs: S652-112 and S2P6, NTD-specific mAbs: 4–8, S652-118, N3C, and 5–7, RBD-specific mAbs B1-182, CB6, A20-29.1, A19-46.1, LY-COV555, A19-61.1, S309, A23-97.1, A19-30.1, A23-80.1, and CR3022, and SD1-specific mAb A19-36.1. Negative control antibody or competitor mAb was injected over both active and reference surfaces.
For analysis, sensorgrams were aligned to Y (Response Units) = 0, using Biacore 8K Insights Evaluation Software (Cytiva) beginning at the serum association phase. Relative “analyte binding late” report points (RU) were collected and used to calculate percent competition (% C) using the following formula: % C = [1 – (100 ∗ ( (RU in presence of competitor mAb) / (RU in presence of negative control mAb) ))]. Results are reported as percent competition.
For analysis, sensorgrams were aligned to Y (Response Units) = 0, using Biacore 8K Insights Evaluation Software (Cytiva) beginning at the serum association phase. Relative “analyte binding late” report points (RU) were collected and used to calculate percent competition (% C) using the following formula: % C = [1 – (100 ∗ ( (RU in presence of competitor mAb) / (RU in presence of negative control mAb) ))]. Results are reported as percent competition.
9
SPR Analysis of IL12Rβ2-IL12 Interaction
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His-tagged Fc-fused human IL12Rβ2 (R&D systems, catalog no. 1959-B2B) and wild-type recombinant human IL12 (rhIL12; R&D Systems, catalog no.219-IL/CF) were used for surface plasmon resonance (SPR) analysis. Anti-His Tag antibody (Cytiva his capture kit, catalog no. 28995056) was diluted to 25 μg/mL in 10 mmol/L sodium acetate pH 4.5 (Cytiva, catalog no. BR100350) and then immobilized onto a CM5 SPR sensor chip (Cytiva, catalog no.BR100530) via amine coupling using EDC/NHS chemistry. His-tagged human IL12Rβ2 was diluted to 2.5 μg/mL in running buffer (1x HBS-EP: 0.01 mol/L HEPES, pH 7.4, 0.15 mol/L NaCl, 0.05% v/v Surfactant P20) supplemented with an additional 0.15 mol/L NaCl) and then was captured onto an anti-his sensor surface. XTX301, proteolytically cleaved XTX301, and rhIL12 were flowed over the chip at concentrations of 3.125 nmol/L to 400 nmol/L with 2-fold dilution. Each concentration of analyte was flowed over the captured ligands for 60 seconds and then a running buffer was flowed for 120 seconds to measure the dissociation of the analyte from the ligands. To remove any residual ligand and/or analyte post-dissociation, the chips were regenerated with 10 mmol/L Glycine pH 1.5 (Cytiva, catalog no. BR100354).
10
Binding Kinetics of Immune Checkpoint Receptors
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Binding kinetics of vdPVR-Fc, mutant vdPVR-Fc and PVR-Fc (786608; Biolegend) proteins to mTIGIT-Fc-his (771808; Biolegend), mCD226-his (50232-M08H; SinoBiological) and mCD96-his (788806; Biolegend) were derived from single-cycle kinetic analyses by surface plasmon resonance (SPR) using a Biacore T200 (Cytiva). Briefly, anti-histidine antibodies were immobilized on a CM5 chip (29149604; Cytiva) using the His capture kit (28995056; Cytiva) following manufacturer’s protocol. The target proteins (mTIGIT-Fc-His tag, mCD226-His tag and mCD96-His tag were captured on the anti-histidine coated chip by flowing 1–5 μg/mL of protein at a flow rate of 30 µL/min. Five concentrations (1:2 serial dilutions) of vdPVR-Fc, mutant vdPVR-Fc and PVR-Fc were flown over the immobilized proteins. All proteins were diluted in HBS-EP running buffer (20 mM of HEPES pH 7.4, 150 mM NaCl, 0.005% Tween-20, 3.4 mM EDTA). The derived sensorgrams were fitted to a 1:1 Langmuir binding model and analyzed using the Biacore T200 evaluation software to calculate the on-rate (ka), off-rate (kd), and the equilibrium constant (KD).
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