Affinities of DARPins were measured on a Biacore X100 instrument (GE Healthcare) with HBS‐EP+ running buffer (GE Healthcare). Recombinant CD23 (Uniprot P06734, amino acids 48‐321) was immobilized on a CM5 biosensor chip using a standard amine coupling kit (GE Healthcare) to an immobilization level of 2400 response units (RU). Binding of anti‐CD23 DARPins was assessed in a twofold serial dilution with at least ten concentrations. Samples were injected for 2 min, followed by 10 min dissociation. Kinetic parameters were calculated with BIAevaluation software (GE Healthcare) using global fitting of the binding data with a 1:1 Langmuir binding.
Standard amine coupling kit
Manufactured by GE Healthcare
Sourced in United States
The Standard Amine Coupling Kit is a lab equipment product designed for the immobilization of amine-containing compounds onto activated surfaces. It provides the necessary reagents and buffers to facilitate the covalent attachment of amine-bearing molecules, such as proteins, peptides, or other biomolecules, to support matrices or sensor surfaces. The kit includes pre-measured and pre-tested components to streamline the coupling process and enable reproducible results.
Automatically generated - may contain errors
Lab products found in correlation
Cm5 sensor chip, by GE Healthcare (2 mentions)
Hbs ep running buffer, by GE Healthcare (1 mentions)
Biaevaluation software, by GE Healthcare (1 mentions)
Biacore x100, by GE Healthcare (1 mentions)
Biaevaluation software, by Cytiva (1 mentions)
Biacore 3000, by GE Healthcare (1 mentions)
Hbs p, by GE Healthcare (1 mentions)
Biacore 3000 instrument, by GE Healthcare (1 mentions)
Streptavidin, by Merck Group (1 mentions)
Biacore t100 evaluation software, by GE Healthcare (1 mentions)
Biacore t100 system, by GE Healthcare (1 mentions)
Pierce fab preparation kit, by Thermo Fisher Scientific (1 mentions)
5 protocols using standard amine coupling kit
1
Measuring DARPins Binding to CD23
2
Binding Kinetics Measurement by BIAcore
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EXAMPLE 5
Binding Assay by BIAcore
All surface plasmon resonance measurements were performed on a BIAcore 3000 instrument (GE Healthcare Biosciences AB, Sweden) at 25° C. Running and dilution buffer was PBS (1 mM KH2PO4, 10 mM Na2HPO4, 105 mM NaCl, 2.7 mM KCl), pH 6.0, 0.005% (v/v) Tween 20. Soluble protein A was diluted in 10 mM sodium acetate buffer, pH 5.0, and immobilized on a CM5 biosensor chip using the standard amine coupling kit (GE Healthcare Biosciences AB, Sweden) to obtain protein A surface densities of approximately 1000 RU. HBS-P (10 mM HEPES, pH 7.4, 118 mM NaCl, 0.005% surfactant P20; GE Healthcare Biosciences AB, Sweden) was used as running buffer during immobilization. The Fc- and antibody-conjugates in question were diluted with PBS, 0.005% (v/v) Tween 20, pH 6.0 to a concentration of 450 nM and injected over 3 minutes at a flow rate of 30 μl/minute. Then the soluble ligand was diluted into the same buffer to different concentrations between 70 and 680 nM and injected over 3 minutes at a flow rate of 30 μl/minute. Afterwards the sensor chip was regenerated for 1 minute with PBS, pH 8.0, 0.005% (v/v) Tween 20. Data analysis was performed with the BIAevaluation software (BIAcore, Sweden) (see
3
ALIX-Syntenin-1 Binding Kinetics
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The interaction of ALIX and syntenin-1 was quantified using a BIAcore 3000 instrument (GE Healthcare) in 10 mM HEPES (pH 7.4) buffer, 150 mM NaCl and 0.005% Surfactant P20. Streptavidin (Sigma-Aldrich, St. Louis, MO, USA) was covalently coupled to CM5 sensor chips (GE Healthcare) via primary amines using the standard amine coupling kit (GE Healthcare). For coupling, Streptavidin was injected at 0.5 mg·mL−1 in 10 mM sodium acetate (pH 5.5) with coupling levels ranging from 6000 to 8000 RU. Biotinylated syntenin-1 N-terminal 11-mer peptides (LifeTein LLC, Somerset, NJ, USA) were immobilized at the indicated levels by injection at 20 to 50 mg·mL−1 for 0.5 min over Streptavidin-coupled surfaces. Equilibrium affinity measurements involved injecting ALIX V-domain (purified as described91 (link)) over immobilized syntenin N-terminal peptides at 20 μl/min. Data was collected from two replicates and analysed using Scrubber 2 software (Biosensor Tools LLC, Salt Lake City, UT, USA).
4
Surface Plasmon Resonance Analysis of Sm/RNP Binding
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SPR analysis was done using a BIAcore T100 system (GE Healthcare). 14 µM CD72a CTLDc/s and 15 µM CD72c CTLDc/s recombinant proteins in 10 mM 2-(N-morpholino) ethanesulfonic acid, pH 6.0, were covalently immobilized on a CM5 sensor chip (GE Healthcare) to the level of more than 5,000 resonance units by amine coupling methods using a standard amine coupling kit (GE Healthcare). A control surface was prepared by activating and blocking a CM5 sensor tip with 1 M ethanolamine without protein. Various concentrations of Sm/RNP were injected over sensor chips at a flow rate of 30 µl/min. An association step of 120 s was followed by a dissociation step of 120 s. Responses to Sm/RNP were corrected by the responses with a control surface and those from a buffer only injection. Data were analyzed by BIAcore T100 Evaluation software (version 2.0.2; GE Healthcare) using a 1:1 binding model.
5
Characterizing Antibody-Antigen Interactions
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Binding experiments were carried out using the Biacore T200 instrument and the Control software 2.0.1. A standard amine coupling kit (GE HEALTHCARE, USA) was used for covalent attachment of 4 ug/mL of erEGF-R recombinant protein to a CM5 biosensor chip in 10 mmol/L sodium acetate buffer at pH 4.5. The immobilized ligand reached a final response of 783.3 RU.
Fab fragments from each antibody to be tested were obtained through papain digestion using the Pierce Fab Preparation Kit (THERMO SCIENTIFIC). HBS-EP+ at pH 7.4 (GE HEALTHCARE) was used as running buffer for multiple cycle analysis of analyte binding at different concentrations of each purified Fab (derived from nimotuzumab, K4 mAb or K5 mAb), in the range between 2.5 and 600 nmol/L. Each sample was injected during 300 s at a flow rate of 15 µL/min. The surface was regenerated with 10 mmol/L of Gly-HCl, pH 2.5. Sensorgrams were analyzed using Biacore T200 evaluation software 3.0. Kinetic data were globally fitted to the 1:1 model.
Fab fragments from each antibody to be tested were obtained through papain digestion using the Pierce Fab Preparation Kit (THERMO SCIENTIFIC). HBS-EP+ at pH 7.4 (GE HEALTHCARE) was used as running buffer for multiple cycle analysis of analyte binding at different concentrations of each purified Fab (derived from nimotuzumab, K4 mAb or K5 mAb), in the range between 2.5 and 600 nmol/L. Each sample was injected during 300 s at a flow rate of 15 µL/min. The surface was regenerated with 10 mmol/L of Gly-HCl, pH 2.5. Sensorgrams were analyzed using Biacore T200 evaluation software 3.0. Kinetic data were globally fitted to the 1:1 model.
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