All surface plasma resonance assays were performed on a Biacore 3000 (GE Healthcare) with a running buffer of 10 mM HEPES pH 7.5 and 150 mM NaCl, supplemented with 0.05% Tween 20 at 25°C. Initial epitope mapping was performed by the binding of SARS-CoV RBD (residue 306–577) and other SARS-CoV-2 antigens (S1 and S2 obtained from BEI Resources) to immobilized CV3-13 IgG (~5800 RU) on a Protein A sensor chip (Cytiva). For the kinetic measurement of CV3-13 Fab binding to SARS-CoV-2 spike, ~800 RU of his-tagged SARS-CoV-2 S-6P was immobilized on a Ni-pretreated NTA chip (Cytiva). 2-fold serial dilutions of purified CV3-13 Fab were then injected with concentrations ranging from 3.125 to 200 nM. Sensorgrams were corrected by subtraction of the corresponding blank channel as well as for the buffer background and kinetic constants determined using a 1:1 Langmuir model with the BIAevaluation software (GE Healthcare). Goodness of fit of the curve was evaluated by the Chi2 of the fit with a value below 3 considered acceptable.
Ni pretreated nta chip
Manufactured by Cytiva
The Ni-pretreated NTA chip is a laboratory equipment designed for protein purification. It features a nickel-nitrilotriacetic acid (Ni-NTA) surface that can bind to proteins with a histidine tag. This chip is used to capture and purify target proteins from complex mixtures in a controlled and efficient manner.
Automatically generated - may contain errors
2 protocols using ni pretreated nta chip
1
Kinetic Binding Analysis of CV3-13 Fab
2
Kinetic Analysis of CV3-13 Fab Binding to SARS-CoV-2 Spike
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All surface plasma resonance assays were performed on a Biacore 3000 (GE Healthcare) with a running buffer of 10 mM HEPES pH 7.5 and 150 mM NaCl, supplemented with 0.05% Tween 20 at 25°C. Initial epitope mapping was performed by the binding of SARS-CoV RBD (residue 306-577) and other SARS-CoV-2 antigens (S1 and S2 obtained from BEI Resources) to immobilized CV3-13 IgG (∼5800 RU) on a Protein A sensor chip (Cytiva). For the kinetic measurement of CV3-13 Fab binding to SARS-CoV-2 spike, ∼800 RU of his-tagged SARS-CoV-2 S-6P was immobilized on a Ni-pretreated NTA chip (Cytiva). 2-fold serial dilutions of purified CV3-13 Fab were then injected with concentrations ranging from 3.125 to 200 nM. Sensorgrams were corrected by subtraction of the corresponding blank channel as well as for the buffer background and kinetic constants determined using a 1:1 Langmuir model with the BIAevaluation software (GE Healthcare). Goodness of fit of the curve was evaluated by the Chi2 of the fit with a value below 3 considered acceptable.
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