SPR assays were used to assess the peptides 1907 and 1907-B binding affinities of human serum albumin (HSA) via Biacore 8K, a high-sensitivity SPR system (Cytiva, UK). Following the standard amine coupling protocol, HSA was overlaid on the sensor. 1907 or 1907-B was dissolved respectively to a series of concentrations in running buffer (PBS, pH = 7.4, Sigma–Aldrich Ltd., Merck, Germany). Samples were permitted to flow through the chip surface at the flow rate of 25 μL/min. After each injection cycle, the surface was injected 25 μL of 10 mmol/L NaOH (Sigma–Aldrich Ltd., Merck, Germany) twice for regeneration. The data were fit by Biacore 8K analysis software (Cytiva) to calculate the binding constants of 1907 or 1907-B.
Biacore 8k
Manufactured by Cytiva
Sourced in United States, Sweden, United Kingdom
The Biacore 8K is a label-free real-time interaction analysis system designed for high-throughput screening and characterization of biomolecular interactions. It utilizes surface plasmon resonance (SPR) technology to monitor binding events in real-time without the need for labeling.
Automatically generated - may contain errors
Lab products found in correlation
Insight evaluation software, by Cytiva (5 mentions)
Biacore insight evaluation software, by Cytiva (5 mentions)
Cm5 sensor chip, by Cytiva (4 mentions)
Series s sensor chip cm5, by Cytiva (4 mentions)
Surfactant p20, by Cytiva (2 mentions)
Series s sa, by Cytiva (2 mentions)
Hbs p buffer, by Cytiva (2 mentions)
Sodium hydroxide (naoh), by Merck Group (2 mentions)
Phosphate buffered saline (pbs), by Merck Group (2 mentions)
Proteon xpr36 protein interaction array system, by Bio-Rad (2 mentions)
Biacore evaluation software, by Cytiva (2 mentions)
Biacore t200, by Cytiva (2 mentions)
Biaevaluation version 4, by GE Healthcare (1 mentions)
Avitag, by Avidity (1 mentions)
Biacore 8k evaluation software 3, by Cytiva (1 mentions)
Cm5 chip, by GE Healthcare (1 mentions)
Surfactant p20, by GE Healthcare (1 mentions)
Neutravidin, by Thermo Fisher Scientific (1 mentions)
Cm5 biosensor chip, by GE Healthcare (1 mentions)
Biacore x100, by Cytiva (1 mentions)
48 protocols using biacore 8k
1
Peptide Binding Affinity Measurement
2
Peptide Binding Affinity Measurement
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SPR assays were used to assess the peptides 1907 and 1907-B binding affinities of human serum albumin (HSA) via Biacore 8K, a high-sensitivity SPR system (Cytiva, UK). Following the standard amine coupling protocol, HSA was overlaid on the sensor. 1907 or 1907-B was dissolved respectively to a series of concentrations in running buffer (PBS, pH = 7.4, Sigma–Aldrich Ltd., Merck, Germany). Samples were permitted to flow through the chip surface at the flow rate of 25 μL/min. After each injection cycle, the surface was injected 25 μL of 10 mmol/L NaOH (Sigma–Aldrich Ltd., Merck, Germany) twice for regeneration. The data were fit by Biacore 8K analysis software (Cytiva) to calculate the binding constants of 1907 or 1907-B.
3
Kinetic Analysis of RBD Protein Binding
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The SPR assays were carried out using a BIAcore 8k (Cytiva) at 25°C. The buffer for proteins used for kinetic analyses were PBST (10mM Na2HPO4, 2 mM KH2PO4 pH7.4, 137 mM NaCl, 2.7mM KCl, 0.005% Tween20). Prototype, Delta and Omicron BA.1 monomeric RBD proteins and PPP and PDO RBD-trimers were diluted with sodium acetate pH 5.0 and immobilized on a CM5 chip with the standard EDC/NHS coupling method. Gradient concentrations of hACE2 protein and antibody’s fab proteins were prepared and flowed over the chip surface with single cycle method. Data were collected over time and 10 mM Glycine pH 1.5 was used to regenerate the sensor. The binding kinetics (binding affinity, KD) were analyzed using 1:1 binding model with the software BIAevaluation Version 4.1 (GE Healthcare).
4
Affibody Binding Kinetics to HER3 by SPR
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Surface plasmon resonance (SPR) analysis by Biacore 8 K (Cytiva, Marlborough, MA) was performed at 25 °C in a run buffer of HBS-EP (0.01 M HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.005% v/v Surfactant P20, Cytiva) and with 15 mM HCl as regeneration solution. Recombinant human ErbB3/Her3 Fc (R&D Systems, Minneapolis, MN) was immobilized by standard amine coupling (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS)) on a CM5 sensor chip (Cytiva, Marlborough, MA) at ~ 1000 RU. The coated chip was preconditioned by three regeneration rounds to stabilize surfaces prior to injection of analyte. Binding of Affibody molecules to HER3 was analyzed by single cycle kinetics via injection of analyte at five different concentrations of purified Affibody molecule (1.25, 2.5, 5, 10 and 20 nM) over immobilized HER3/Fc. The experiment was performed in duplicates. Biacore Insight Evaluation Software was used to process, analyze and fit data.
5
Surface Plasmon Resonance Binding Assay
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SPR experiments were performed with a Biacore 8K instrument (Cytiva) using PBST. For binding assays of AiDs with HA-PD1, HA-PD1 with a C-terminal AviTag was biotinylated with BirA according to the manufacturer’s protocol (Avidity) and immobilized on a NeutrAvidin chip (Cytiva, 29407997) to a level of ~250 response units (RUs). Binding measurements were performed using single-cycle kinetics with five 1:3 serial dilutions of each recombinant AiD (at concentrations indicated in figures) using 120 seconds of association and 1200 seconds for the final dissociation time at 25°C and a flow rate of 30 μL/min. The data were analyzed in the same manner as the masked antagonists.
6
SPR Assay for FKBP12 Binding Affinity
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Example 28
SPR Assay to Determine Binding Affinity to FKBP12.
Biotinylated avi-FKBP12 was immobilized on a streptavidin chip (Cytiva Series S SA) using a Biacore 8K or 8 k+ (Cytiva). To achieve an immobilization level of 1000 RU, 2 μg/ml biotinylated avi-FKBP12 were injected for 100 sec at a flow rate of 10 μl/min. Rapalogs were diluted in DMSO to 100× working concentration. Each Rapalog was 100-fold diluted in 50 mM HEPES pH 7.5, 150 mM NaCl, 2 mM MgCl2, 1 mM DTT, 0.05% Tween-20 and a serial dilution prepared (9 concentrations, 3-fold dilutions, 0.08-500 nM). Rapamycin was used as reference sample (9 concentrations, 3-fold dilutions, 0.02-100 nM). The compound dilutions were then injected at 100 uL/min for 120 seconds contact time in sequence with increasing concentrations. Dissociation was monitored for 3600 seconds. 50 mM HEPES pH 7.5, 150 mM NaCl, 2 mM MgCl2, 1 mM DTT, 0.05% Tween-20, 1% DMSO was used as running buffer. The single-cycle kinetics data were fit to a 1:1 binding model to measure the association rate ka (1/Ms), the dissociation rate kd (1/s) and the affinity Kd (M). Table 8 includes FKBP12 direct binding Kd (nM) values of selected compounds; with compounds having a FKBP12 direct binding Kd of less than 0.3 nM as A, 0.3 nM to 1.0 nM as B, and greater than 1.0 nM as C.
7
Kinetic Analysis of Stx2a-P-stalk Interaction
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The Biacore 8K+ (Cytiva) was used to study the interaction. Stx2a was immobilized on Fc2 of a CM5 chip in four different channels using amine coupling. Fc1 was activated and blocked. The P-stalk was flown over the surface of the chip at different concentrations listed in Figures 2 E and S1 B, at a flow rate of 30 μl/min, for 30 s and then left to dissociate for additional 60 s. The running buffer contained 10 mM Hepes (pH 7.4), 150 mM NaCl, 10 mM MgCl2, 3 mM EDTA, and 0.05% P20. Because of the fast on/off kinetics, the dissociation constants (KD) for the interactions were determined by fitting the binding data at steady state using Biacore Insight Evaluation Software 3.0 (Figs. 2 F and S1 C).
8
Binding Kinetics of SARS-CoV-2 Variants to ACE2
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SPR experiments were performed on the Biacore 8K (Cytiva). Human ACE2 with Fc tag was immobilized onto Protein A sensor chips (Cytiva). Purified SARS-CoV-2 variant RBDs were prepared in serial dilutions (6.25, 12.5, 25, 50, and 100nM) and injected over the sensor chips. The response units were recorded by Biacore 8K Evaluation Software 3.0 (Cytiva) at room temperature, and the raw data curves were fitted to a 1:1 binding model using Biacore 8K Evaluation Software 3.0 (Cytiva).
9
SPR Analysis of Protein-Ligand Interactions
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SPR measurements were performed on the Biacore 8K (Cytiva; v.4.0.8.19879) system with HBS-EP+ as the running buffer (10 mM HEPES pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.005% (v/v) Surfactant P20, GE Healthcare). Ligands were immobilized on the CM5 chip (GE Healthcare, 29104988) through amine coupling. In total, 500–1,000 response units (RU) were immobilized and designed proteins were injected as an analyte in serial dilutions. The flow rate was 30 µl min−1 for a contact time of 120 s followed by 800 s dissociation time. After each injection, the surface was regenerated using 3 M magnesium chloride (for PD-L1) or 10 mM glycine, pH 3.0 (for RBD). Data were fit with a 1:1 Langmuir binding model within the Biacore 8K analysis software (Cytiva, v.4.0.8.19879).
10
Measuring RNA Aptamer Binding Kinetics
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CM5 sensor chips (Cytiva Life Sciences, Marlborough, MA) were prepared by amine coupling of NeutrAvidin (ThermoFisher, Waltham, MA). The RNA aptamer was modified by inclusion of biotin and a (dA)10 spacer at the 3′ end. The running buffer contained 150 mM NaCl, 5 mM MgCl2, 10 mM HEPES (pH 7.4), and 0.01–0.1% DMSO. The RNA was present at 2 µg/mL concentration and was heated at 95°C for 2 min and then quickly cooled on ice. The ligand was added at a concentration of 0 to 0.1–2.0 µM (depending on affinity) and binding data were obtained using a Biacore 8K or T200 surface plasmon resonance system (Cytiva Life Sciences), run at 25°C at a flow rate of 30 µL/min.
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