Experiments were performed on a Biacore X100 system (Biacore AB, Uppsala, Sweden). Recombinant human NQO1 and GSTP1 dissolved in 10 mM sodium acetate buffer (pH = 5.0) were covalently immobilized in the dextran matrix of a CM5 sensor chip with the Amine Coupling Kit using a standard primary amine coupling procedure. The compound MNPC was injected into the flow cells in running buffer at a flow rate of 30 μL/min for 120 s of association phase, followed by a 120-s dissociation phase and a 30-s regeneration phase. The surface of the sensor chip was regenerated via the injection of 10 μL of the regeneration buffer (5 mM NaOH). The association rate constant ka and dissociation rate constant kd were calculated and analyzed using the monovalent analyte model, and the equilibrium dissociation constant (KD) was calculated (KD = kd/ka) [29 ].
Biacore x100 system
Manufactured by Cytiva
Sourced in Sweden
The BIAcore X100 system is a label-free biosensor designed for biomolecular interaction analysis. It utilizes surface plasmon resonance (SPR) technology to monitor real-time binding events between molecules without the need for labeling. The system provides quantitative information on kinetics, affinity, and concentration of biomolecular interactions.
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Lab products found in correlation
Biacore x100 evaluation software, by Cytiva (2 mentions)
Amine coupling kit, by GE Healthcare (2 mentions)
Cm5 chip, by Cytiva (2 mentions)
Biacore x100, by Cytiva (2 mentions)
Superdex peptide, by GE Healthcare (1 mentions)
Surfactant p20, by GE Healthcare (1 mentions)
Biotin capture kit, by Cytiva (1 mentions)
Avitag, by ACROBiosystems (1 mentions)
Hbs ep ph 7, by Cytiva (1 mentions)
Polysorbate 20, by AppliChem (1 mentions)
Human antibody capture kit, by Cytiva (1 mentions)
Sodium chloride (nacl), by Carl Roth (1 mentions)
Sodium phosphate, by Carl Roth (1 mentions)
Cm5 sensor, by Cytiva (1 mentions)
Potassium chloride (kcl), by Carl Roth (1 mentions)
Anti gst antibody, by Cytiva (1 mentions)
Cm5 sensor chip, by Cytiva (1 mentions)
Sensor chip nta, by Cytiva (1 mentions)
Avanti mini extruder, by Avanti Polar Lipids (1 mentions)
11 protocols using biacore x100 system
1
Kinetic Analysis of NQO1 and GSTP1 Binding
2
Surface Plasmon Resonance of Heparin-PNA-Fc Binding
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Surface plasmon resonance (SPR) experiment was performed with the Biacore X100 system and Biacore X100 evaluation software (Biacore AB, Uppsala, Sweden). Heparin was coupled with biotin-hydrazide (Dojindo, Kumamoto, Japan) by reductive amination. Briefly, 1 mg of heparin solubilized in 125 μl of 0.1 M 2-(N-morpholino)ethanesulfonic acid (MES), pH 5.5, was mixed with 250 μl of 72 mM biotin-hydrazide in dimethyl sulfoxide and 250 μl of 1 M sodium cyanoborohydride in 0.1 M MES, pH 5.5. After the mixture was allowed to stand at 20°C for 12 h, biotinylated heparin was purified on a column of Superdex peptide (3 × 250 mm, GE Healthcare). Biotinylated heparin was immobilized to streptavidin-coupled sensor chip SA (Biacore) until the resonance units reached approximately saturation (approximately 130 resonance unit (RU)). The binding of mutated PNA-Fc to heparin was measured in 10 mM HEPES, pH 7.4, containing 150 mM NaCl and 0.005% surfactant P20 (GE Healthcare) at 25°C with a flow rate of 20 μl/min. The mutated PNA-Fc at 1, 0.2, 0.04, 0.008, and 0.0016 μM was applied to the sensor chip. The regeneration of the chip surface was carried out with 50 mM glycine-HCl, pH 3.0, containing 150 mM NaCl and 0.005% surfactant P20.
3
SARS-CoV-2 RBD-ACE2 Binding Kinetics
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The measurements were performed with a Biacore X-100 system and the Biotin CAPture kit (Cytiva). The running buffer was HBS-EP+ pH 7.4 (Cytiva). The ligand SARS-CoV-2 RBD with an AviTag (Acrobiosystems) was captured on the streptavidin chip to around 100 RU. Increasing concentrations of the analyte ACE2-Fc (0.32, 1.6, 8, 40 and 200 nM) were injected over the immobilized ligand in a single-cycle kinetic mode. The obtained sensorgrams were evaluated with the Biacore X-100 software to obtain a binding constant (KD).
4
Quantifying HLA-CLIP-γδ TCR Interactions
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To determine the HLA-CLIP- γδ TCR-binding specificity and affinity constants, surface plasmon resonance was performed with the BIAcore X-100 system (Cytiva; previously GE Healthcare) as described previously (González-Motos et al., 2017 (link)). Experiments were performed at 25°C using HBS-EP (0.01 M Hepes, pH 7.5, 0.15 M NaCl, 3 mM EDTA, 0.05% Tween20) as running buffer. On both flow cells of a CM5 chip (Cytiva) about 1,500 response units (RU) StrepTactinXT (Twin-Strep-tag Capture Kit from iba) were immobilized via amine coupling using acetate buffer, pH 4.5 (amine coupling kit from GE Healthcare). Twin-Strep-tagged HLA-CLIP was captured on FC2 and the TCRs were injected into both flow cells. For binding assays, analytes were injected at 2.5 µM for 90 s followed by 60 s of dissociation with a flow rate of 10 µl/min. For single-cycle kinetics, the contact time was increased to 120 s and the dissociation time to 90 s with a flow rate of 30 µl/min. To regenerate the StrepTactinXT surface, 3-M GuHCl was injected for 60 s. All analyses were performed with the Biacore ×100 Evaluation Software. The sensorgrams of FC2-1 were adjusted and a blank injection was subtracted. For kinetic analyses, data were fitted using a 1:1 binding model.
5
Expression and Purification of DCLK1 Kinase Domain
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The expression and purification of DCLK1 kinase domain were same as those described in the Supplementary Materials in our previous work [7 (link)]. Briefly, DCLK1 kinase domain was overexpressed in Escherichia coli strain Rosetta (DE3) (Novagen, Darmstadt, Germany) and purified sequentially by nickel affinity chromatography and gel filtration chromatography. SPR experiments were performed using a Biacore X-100 system (Cytiva, Washington, DC, USA) at room temperature, with a running buffer of 10 mM Na2HPO4, 1.75 mM KH2PO4, pH 7.2~7.6, supplemented with 0.137 M NaCl, 2.65 mM KCl, 0.05% Tween-20, and 5% DMSO. DCLK1 kinase domain (KD) was covalently immobilized onto a CM5 sensor chip using the Amine coupling kit (GE Healthcare). The immobilization of KD resulted in ~12,000 response units (RU). Small-molecule inhibitors were injected over the flow cells at a range of eight concentrations prepared by serial two-fold dilutions, at a flow rate of 30 μL min−1, with an association time of 120 s and a dissociation time of 180 s. The data were fitted to a 1:1 binding model so as to calculate equilibrium dissociation constants (binding affinity, KD) using GraphPad Prism 8.
6
Surface Plasmon Resonance Analysis of CD123 Binding Kinetics
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Binding kinetics of TM123-4-1BBL and TM123 on human CD123 were analyzed by surface plasmon resonance (SRP) using a Biacore® X100 system (Cytiva). Three different and independent batches of purified TM123-4-1BBL and TM123 were included. As mobile phase 20 mM Sodium phosphate, 2.7 mM KCl, 137 mM NaCl (all Carl Roth GmbH + Co. KG), 0.05% (v/v) Polysorbate 20 (AppliChem) in 1000 ml ddH2O pH 7.4 was used. Therefore, the recombinant fragment crystallizable (Fc)-tagged soluble human CD123 was transiently immobilized on a CM5-sensor (Cytiva) containing a covalently bound anti-human capture antibody (Cytiva, Human antibody capture kit). Consequently, decreasing concentrations of TMs were injected in duplicates for 180 seconds allowing analyte association, followed by a 1200 second dissociation phase. With independent cycles, different TM concentrations ranging from 50 nM to 0.62 nM were tested; in between, the biosensor was regenerated using 3 M Magnesium chloride (Cytiva, Human antibody capture kit). The measured data is displayed in response units (RU) and analyzed using the Biacore® X100 software (Cytiva). The raw data were fitted using a monovalent 1:1 kinetic binding model, and the bulk signal was set constant to zero (RI = 0).
7
Influenza Hemagglutinin Binding Kinetics
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The direct interaction between HBL40 and the influenza viral envelope glycoprotein hemagglutinin was analyzed by surface plasmon resonance (SPR) on a BIAcore X100 system (Cytiva, Tokyo, Japan), as previously described [10 (link),11 (link),12 (link),13 (link)]. The influenza vaccine preparation (Denka-Seken), which contains a mixture of the hemagglutinin proteins of A/California/7/09 (H1N1), A/Victoria/210/09 (H3N2), and B/Brisbane/60/08, was immobilized as the viral hemagglutinin onto the sensor chip, which was activated with N-hydroxysuccinimide/N-ethyl-N′-dimethylaminopropyl carbodiimide. Various concentrations of lectin solution were used for binding experiments with a running buffer of HBS-N consisting of 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 150 mM NaCl (pH 7.4) at a flow rate of 30 μL/min. The contact time and dissociation time were performed as 120 s and 600 s, respectively. Kinetic parameters (ka, kd, KA, and KD) were calculated by fitting the data to the Langmuir model for 1:1 binding using the Biacore X100 evaluation software (Cytiva, Tokyo, Japan).
8
Kinetic Analysis of mAb521-SlpA Interaction
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Prior to SPR, mAb521 and SlpA were dialysed extensively into PBS. All analyses of interactions between mAb521 and SlpA were performed on a BIAcore X100 system equipped with a CM5 chip (BIAcore GE Healthcare) at a flow rate of 10 μl/min. For immobilization of SlpA, the chip was activated with NHS/EDC (1:1) before injection of SlpA in 10 mM acetate, pH 4.0 to attain 255 Resonance Units (RU) of binding. This is designated the FC2 sample channel. In contrast the FC1 reference channel was NHS/EDC treated and then blocked using ethanolamine. Monoclonal antibody 521 (250 nM -0.65 nM) was injected over both reference and sample surfaces for 300 s before a 600 s wait time followed by a 12 s regeneration using 10 mM glycine pH 2.0. Injection of PBS alone served as a negative control. All measurements were performed in duplicate at 25°C in PBS. The results derived from the reference channel FC1 were subtracted from the FC2 sample channel. SPR data was exported and plotted using OriginPro 8. The responses for each of the different antibody concentrations at 380 s were taken and fitted to a sigmoidal fitting function to derive the apparent KD.
9
Quantifying Protein-Protein Interactions Using SPR
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Interactions between recombinant h4.1R (h4.1R WT and h4.1R[PE14]) and GST-hNβSp were analyzed by measuring surface plasmon resonance on the Biacore X100 system (Cytiva). GST-hNβSp or GST alone as the control, at concentrations of 500 nM, was immobilized on the surface of CM5 sensor chips (Cytiva) using an anti-GST antibody (MBL). Association and dissociation analyses of recombinant proteins at appropriate concentrations (150–1000 nM) were performed in PBS at 25 °C. At the end of each measurement, the chip was regenerated in 10 mM Glycine–Cl (pH 2.1). Data were processed with the Biacore X100 evaluation software (biacore-x100-software">https://www.cytivalifesciences.com/en/us/support/software/biacore-downloads/biacore-x100-software , Cytiva).
10
Characterizing ColN-LPS Interactions via BIAcore
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All analyses of interactions between ColN domain mutants and Rc/Rd LPS were performed on a BIAcore X100 system equipped with a NTA sensor chip (Biacore GE Healthcare) at a flow rate of 5 μl min−1. For immobilization of proteins, the chip was treated with 500 μM NiCl2 for 1 min before a 60 s injection of purified protein (500 nM). Then 100 μM Rc or Rd LPS was injected for 60 s and the dissociation followed. In order to continuously monitor the non‐specific background binding of samples to the Ni2+ surface, LPS was injected over a control flow cell which lacked protein. Regeneration of the chip surface required a 180 s injection of 10 mM HEPES pH 8.3, 150 mM NaCL, 350 mM EDTA followed by a 60 s injection of 100 mM NaOH. All measurements were performed at 25°C in buffer containing 10 mM HEPES pH 7.5, 150 mM NaCl, 50 μM EDTA. Data were analysed with BIAcore X100 evaluation software ver.1.0 (Biacore GE Healthcare). In all cases results presented are representative of a minimum of triplicate experiments.
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