Apparent binding affinity constants were determined using surface plasmon resonance on a Biacore X100 instrument (GE Healthcare). The complementary primer sequences (Supplementary methods), of which one was functionalized with biotin on the 5′ end, were combined and diluted in HBS-P buffer (10 mM HEPES pH 7.4, 0.15 M NaCl, 0.005% Surfactant P20). The DNA substrate was immobilized in one of two flow channels on a streptavidin coated sensor chip SA (GE Healthcare). Kuwt and Kucore were diluted in HBS-P buffer and their association to DNA was recorded by injecting protein at a flow rate of 30 μl/min for 180 s. The dissociation phase was observed for 600 s by flowing HBS-P buffer followed by surface regeneration using 0.2 M NaOH. Triplicates were run for each concentration and the responses were background corrected by subtracting the signal from the non-modified flow channel. The resulting sensograms were fitted to the standard kinetic interaction model provided by the Biacore system for interaction of an analyte with a heterogeneous ligand.
Biacore x100 instrument
Manufactured by GE Healthcare
Sourced in Sweden, United States
The Biacore X100 is a label-free, real-time interaction analysis instrument. It is designed for studying biomolecular interactions, such as protein-protein, protein-small molecule, and protein-DNA interactions. The instrument uses surface plasmon resonance (SPR) technology to detect and measure these interactions in real-time without the need for labeling.
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Lab products found in correlation
Biacore x100, by Cytiva (32 mentions)
Cm5 sensor chip, by GE Healthcare (10 mentions)
Biacore x100 evaluation software, by GE Healthcare (6 mentions)
Biacore x100 evaluation software version 2, by GE Healthcare (3 mentions)
Biaevaluation software 2, by GE Healthcare (2 mentions)
Human antibody capture kit, by GE Healthcare (2 mentions)
Biacore reagents and software, by GE Healthcare (2 mentions)
Prism 8, by GraphPad (2 mentions)
Biotin capture kit, by GE Healthcare (2 mentions)
Biacore x100 control software version 2, by GE Healthcare (2 mentions)
Bia evaluation 3.1 analysis software, by GE Healthcare (2 mentions)
Amine coupling kit, by GE Healthcare (2 mentions)
Nta sensor chip, by GE Healthcare (2 mentions)
Nta reagent kit, by GE Healthcare (2 mentions)
Streptavidin coated sa sensor chip, by GE Healthcare (1 mentions)
Biacore cm5 sensor chip, by GE Healthcare (1 mentions)
Cm5 chip, by GE Healthcare (1 mentions)
Series s sensor chip cm5, by Cytiva (1 mentions)
Megashortscript t7 kit, by Thermo Fisher Scientific (1 mentions)
N hydroxysuccinimide nhs, by GE Healthcare (1 mentions)
37 protocols using biacore x100 instrument
1
Ku Protein Binding Affinity Analysis
2
Tpm-SMTNL1-TMB Binding Kinetics by SPR
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The binding between Tpm and SMTNL1-TMB with or without phosphorylation was evaluated by SPR using a BIAcore X100 instrument (GE Healthcare). Purified Tpm was immobilized via amine-coupling onto a CM5 sensor chip (GE Healthcare). The running buffer contained 20 mM HEPES pH 7.5, 100 mM KCl, 1 mM DTT, and 0.005% (v/v) Tween-20. Five concentrations of SMTNL1-TMB from 0.12 to 10 μM were prepared by serial dilution and were injected at a flow rate of 30 μL/min with a contact time of 1 min at 25 °C. The chip was regenerated by injecting 1 M NaCl for 4 min followed by glycine-HCl (10 mM, pH 3.0) for 1 min. Each experiment was repeated three times (n = 3) to obtain a standard error (SE). The BIAevaluation software 2.0 (GE Healthcare) was used to analyze the SPR sensorgrams and to obtain the dissociation constants (Kd).
3
Kinetic and Thermodynamic Profiling of Anti-ROR Fabs
4
Kinetics of Aβ42 Protofibrils Binding
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Surface plasmon resonance (SPR) studies were performed on a Biacore X100 instrument (GE Healthcare). The Aβ42cc protofibrils were immobilized onto a Biacore CM5-sensor chip (GE Healthcare), as described previously31 (link).
Five or six concentrations of each analyte were prepared in HBS-EP (10 mM HEPES, 150 mM NaCl, 3 mM ETDA, 0.005% Tween-20, pH 7.4) and injected over the immobilized chip surface for 250 s to record analyte binding to the surface. Dissociation was observed for 2,000 s in running buffer. The sensor surface was regenerated after each injection with 20 mM NaOH with 90 s contact times. All experiments were carried out at 25 °C with a flow rate of 10 μL/min.
SPR data sets were analyzed using Biacore X100 Evaluation 2.0.1 software and curve fitting was performed with a heterogeneous binding site model using global kinetic fitting, but with local adjustment of the parameter Rmax.
Five or six concentrations of each analyte were prepared in HBS-EP (10 mM HEPES, 150 mM NaCl, 3 mM ETDA, 0.005% Tween-20, pH 7.4) and injected over the immobilized chip surface for 250 s to record analyte binding to the surface. Dissociation was observed for 2,000 s in running buffer. The sensor surface was regenerated after each injection with 20 mM NaOH with 90 s contact times. All experiments were carried out at 25 °C with a flow rate of 10 μL/min.
SPR data sets were analyzed using Biacore X100 Evaluation 2.0.1 software and curve fitting was performed with a heterogeneous binding site model using global kinetic fitting, but with local adjustment of the parameter Rmax.
5
Kinetic Analysis of Anti-DARC Nanobodies
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Interactions between the anti-DARC BMFPs and DARC were studied by SPR, using a Biacore X100 instrument (GE Healthcare). All experiments were performed in HBS-EP (Hepes buffer saline EDTA surfactant P20) buffer (GE Healthcare) at 25°C. For studying the binding of Nb-αDARC, Nb-αDARC-P18F2, Nb-αDARC-P18F3, and Nb-αDARC-P18F4 to DARC, DARC-ECD1 was immobilized on the analysis Fc2 channel of a CM5 chip (GE Healthcare) by amine coupling, whereas DARC-ECD1-Mut was immobilized on the reference channel Fc1. Both channels were then blocked with 1 M ethanolamine-HCl (pH 8.5). BMFPs were injected at 30 μl min−1 in dilution series (0.1 to 125 μM) over the coated chips. Between the injections, the chip surface was regenerated with two injections of 15 μl of 10 mM HCl (pH 2.0). The specific binding responses to the molecular targets were obtained by subtracting the response given by the analytes on Fc2 with the response on Fc1. The kinetic sensorgrams were fitted to a global 1:1 interaction Langmuir model, and the koff and kon values were calculated using the manufacturer’s software (Biacore X100 Evaluation version 2.0).
6
Quantifying rTpiA Interactions with Key Proteins
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The molecular basis of the binding of rTpiA to human plasminogen, laminin, and BSA was investigated using surface plasmon resonance (SPR)‐based binding techniques, as previously described [11 (link), 23 (link), 24 (link)], with some modifications. Briefly, SPR measurements were performed using a Biacore X100 instrument (GE Healthcare). rTpiA was diluted with 10 mm sodium acetate buffer (pH 4) to a concentration of 50 μg·mL−1 and immobilized on a Series S Sensor Chip CM5 (Cytiva) using an amine coupling kit according to the manufacturer’s instructions. Human plasminogen, laminin, and BSA were diluted with running buffer (100 mm HEPES pH 7.4, containing 150 mm NaCl, 3 mm EDTA, and 0.005% surfactant P20) and injected into the flow cell. The flow rate was maintained at 10 μL·min−1 for immobilization and at 20 μL·min−1 for analysis. Regeneration of the surface on the sensor chip was achieved by using 50 mm HCl. The data were analyzed using the Biacore X100 evaluation software (GE Healthcare).
7
Measuring ANAM Binding Affinity
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The affinity of the constructed ANAMs for β‐catenin or NF‐κB proteins was measured by surface plasmon resonance using a Biacore X100 instrument (GE Healthcare, Uppsala, Sweden). Briefly, the CM5 sensor chip (GE Healthcare) was preequilibrated with running buffer prior to the immobilization of ligands onto the chip surface and activated with 0.05 M N‐hydroxysuccinimide (NHS; GE Healthcare) and 0.2 M 1‐ethyl‐3‐(3‐dimethylaminopropyl) carbodiimide (EDC; GE Healthcare). Each protein was then injected into the flow cells of the sensor chip, and amine coupling chemistry was used for covalently attaching ligands to the sensor chip surface. After immobilization of the ligand, the chip surface was deactivated with 1 M ethanolamine hydrochloride to block the remaining unreacted groups. DNA template oligonucleotides for ANAMs were transcribed into RNA using the MEGA‐shortscript T7 kit (Life Technologies). Then various concentrations of the ANAMs were injected over the sensor surface for 1.5 min at 5 μL/min, and subsequently analyzed for equilibrium binding properties.
8
Surface Plasmon Resonance Analysis of LL-37 Binding
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LL-37 (10 μm in acetate buffer, pH 5.0; Anaspec) was immobilized onto a CM5 sensor chip (GE Healthcare) using standard amine-coupling at 25°C and targeted at 2000 RU (actual RU 2028) [73 ]. sLL37 (Anaspec) was immobilized on a separate CM5 sensor chip using the same procedure (final signal RU 1726). SPR analyses were performed using a Biacore X100 instrument (GE Healthcare). Running buffer I was HBS-P buffer (GE Healthcare). Running buffer II was composed of 113 mM NaCl, 24 mM NaHCO3, 3.9 mM KCl, 1.3 mM CaCl2, 0.6 mM MgCl2, 0.005% surfactant P20, pH 7.3 [74 (link)]. His-ClnR (as described above for EMSAs) was dialyzed into running buffer II, then diluted into running buffer II for two-fold serial dilutions from 10 μM to 0.625 μM. His-ClnR was injected over immobilized LL-37 or sLL-37 for 180 s association time and 300 s dissociation time. The chip surfaces were regenerated by injecting 1 M NaCl in 50 mM NaOH for 90 s, then the surface was stabilized for 300 s prior to the next cycle run. The flow rate used was 10 μl/min. Binding to the reference cell was subtracted and the data were evaluated by Baicore X100 evaluation software (V2.0.1). Experiments were performed a minimum of two times.
9
Evaluating CIP-C3d Affinity with BIAcore
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The affinity of the interaction between CIP and C3d was evaluated with a BIAcore X100 instrument (GE Healthcare). Human C3d was covalently immobilized on a dextran matrix CM5 sensor chip surface with a C3d solution (30 μg/ml in 50 mM sodium acetate buffer, pH 5) diluted 1:1 with N-hydroxysuccinimide and N‑ethyl‑N′‑(3‑dimethylaminopropyl)carbodiimide hydrochloride. The excess of active groups on the dextran matrix was blocked with 1 M ethanolamine (pH 8.5). On another flow cell, the dextran matrix was treated as previously described but without any ligand to provide an uncoated reference flow cell. The running buffer used was PBS containing 0.005% (v/v) Tween 20. A 2-fold linear dilution series (5 μM up to 0.0390 μM) of CIP in running buffer were passed over the ligand at the flow rate of 45 ml/min, and all the sensorgrams were recorded at 22°C. Surface regeneration was achieved by injecting a solution of 25 mM NaOH. Association and dissociation kinetics parameters (Ka and Kd) and the equilibrium dissociation constant Kd were estimated with a 1:1 interaction model (Langmuir model) by nonlinear fitting, using BIAevaluation 1.0 software (GE Healthcare).
10
SPR Binding Kinetics Analysis
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All binding and kinetics studies were performed on a Biacore X-100 instrument (GE Healthcare) by surface plasma resonance (SPR). The sensor biochip CM5 was activated using EDC/NHS at 10 μL/min for 7 minutes. Goat anti-human IgG Fc mAb was immobilized on two flow cells of the CM5 sensor chip at a concentration of 25μg/ml in 10 mM sodium acetate buffer (pH 5.0) for 7 min at 10 μL/min. HBS–EP was used as the running buffer and blank runs were performed with HBS–EP buffer (or an appropriate buffer for all other experiments). The sample was injected onto the senser chip with a flow rate of 10 μL/min. A series of antigen concentrations including 6.25 nM, 12.5 nM, 25 nM, 50 nM and 100 nM were injected consecutively, each with a contact time of 120 seconds and a dissociation time of 1800 seconds. The chip was regenerated using 3 M magnesium chloride. The datas were analyzed using the BiacoreX100 analytical and evaluation softwares (GE Healthcare). The dissociation rate constant (kd) and the association rate constant (ka) were calculated by a global fitting analysis. The first dissociation equilibrium constant (KD1) was determined from the ratio of the rate constants: KD = kd / ka.
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