Binding and kinetic analyses were performed with 200 to 400 response units of purified recombinant FHbp immobilized to a biosensor chip using amine coupling (CM5 chip and amine coupling kit; GE Life Sciences). The running buffer was HBS-EP (10 mM HEPES [pH 7.4], 150 mM NaCl, 3 mM EDTA, 0.05% surfactant P-20 [GE Life Sciences]); the regeneration buffer was 100 mM glycine (pH 2.0)–3 M NaCl or 10 mM glycine (pH 1.7). For multiple-cycle kinetic experiments, the injection times with the Fabs were 3 min for association, 5 to 10 min for dissociation, and 1 to 2 min for regeneration and the flow rate was 30 µl/min. Data were collected on a Biacore X100 Plus instrument and analyzed with X100 Evaluation software (GE Life Sciences) using a 1:1 binding model.
Biacore x100 plus instrument
Manufactured by GE Healthcare
The Biacore X100 Plus instrument is a label-free biosensor system used for real-time interaction analysis. It is designed to measure and analyze the interactions between biomolecules, such as proteins, peptides, small molecules, and other biological entities. The instrument utilizes surface plasmon resonance (SPR) technology to detect and quantify these interactions without the need for labeling.
Automatically generated - may contain errors
Lab products found in correlation
Surfactant p20, by GE Healthcare (1 mentions)
Hepes, by GE Healthcare (1 mentions)
Ethylene diamine tetraacetic acid (edta), by GE Healthcare (1 mentions)
X100 control software, by Cytiva (1 mentions)
Cm5 chip, by GE Healthcare (1 mentions)
Origin 7, by Malvern Panalytical (1 mentions)
Vp dsc instrument, by Malvern Panalytical (1 mentions)
Human fh, by Complement Technology (1 mentions)
4 protocols using biacore x100 plus instrument
1
Kinetic Analysis of FHbp Binding
2
Kinetics of rEF-Tu and Fibronectin Interaction
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The rEF-Tu and fibronectin interaction dynamics was further investigated in real time by surface plasmon resonance (SPR) experiment on a Biacore™ X100 Plus instrument (GE Healthcare). Fibronectin was diluted to 10 μg/mL in 10 mM sodium acetate (pH 4.0) and covalently linked to the carboxylmethylated dextran matrix of a sensor chip CM5 as ligands using an amine coupling kit (Biacore AB). Immobilization of soluble fibronectin generated resonance units (RU) of 2868. The binding kinetics was measured with the increasing concentrations (0–200 μg/mL) of the analyte (rEF-Tu) in running buffer (HBS-EP + [10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.05% (v/v) surfactant P20]; Biacore AB) at a flow rate of 30 μL/min for 180 s over immobilized fibronectin at 20°C. The dissociation phase was monitored for 1,000 s by allowing buffer to flow over the chip. Association kinetics were analyzed manually using Biacore™ X100 Control Software (Deutscher et al., 2010 (link)).
3
Kinetics of rFBA-Fibronectin Interaction
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The interaction dynamics of rFBA and fibronectin were further investigated in real time by surface plasmon resonance (SPR) using a BIAcore X100 Plus instrument (GE Healthcare). Fibronectin was diluted to 10 µg/mL in 10 mM sodium acetate (pH 4.0) and covalently linked to the carboxymethylated dextran matrix of a CM5 sensor chip as the ligand using an amine coupling kit (Biacore AB). Immobilisation of soluble fibronectin generated resonance units (RU) of 2868. Binding kinetics were measured with increasing concentrations (0–100 μg/mL) of the analyte (rFBA) in running buffer (HBS-EP) consisting of 10 mM HEPES, 150 mM NaCl, 3 mM EDTA, and 0.05% (v/v) surfactant P20 (Biacore AB) at a flow rate of 30 μL/min for 180 s over immobilised fibronectin at 20 °C. The dissociation phase was monitored for 1000 s by allowing the buffer to flow over the chip. Association kinetics were analysed manually using Biacore X100 Control Software [11 (link)].
4
Characterization of FH-FHbp Interaction
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Binding of FH to FHbp was measured in a modified ELISA [22 (link)] using as much as 200 μg/ml of purified human FH. Surface plasmon resonance experiments were performed by immobilizing human FH (Complement Technology; 1500 response units) on a CM5 chip (GE Life Sciences) via amine coupling. Soluble FHbp binding to immobilized FH was measured at concentrations ranging from 3.16 nM to 316 nM using a BIAcore X-100 Plus instrument (GE Life Sciences). Binding of control murine anti-FHbp mAbs to FHbp was measured by ELISA [33 (link)]. We measured the thermal stability of FHbp mutants by differential scanning calorimetry using a VP-DSC instrument (MicroCal), a protein concentration of 0.5 mg/ml and PBS as the diluent and reference solution. The data were collected using a scan rate of 60°C per hour with passive feedback mode and were analyzed using Origin 7.0 Software (MicroCal).
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