Affinity and rates of association were measured on a BIA-core 3000 (GE Healthcare, Hillerod, Denmark). Buffers were sterile filtered and degassed. A nitrilotriacetic acid (NTA) chip (GE Healthcare, Diegem, Belgium) was used to capture histidine-tagged [27 (link)] ligands. The NTA chip was prepared for ligand capturing by injecting NiCl2 (0.5 M) (GE Healthcare, Diegem, Belgium) followed by injection of 20 lL His-ClfA1–520 (200 nM). His-ClfA1–520 and vWbp-Strep were diluted in running buffer (HBS-P buffer (20 mM HEPES [pH 7.4], 150 mM NaCl, 0.005% [vol/vol] surfactant P20) (GE Healthcare, Diegem, Belgium), 50 uM ethylenediaminetetraacetic acid (EDTA) (GE Healthcare, Diegem, Belgium) and 1 mM imidazole). To study the His-ClfA1–520–vWbp interaction, vWbp-Strep was injected at 6.25 nM, 12.5 nM and 25 nM for 180 s, followed by regeneration of the chip with 50 lM EDTA and 1 mM imidazole. All injections were performed at a flow rate of 10 lL min−1. Kinetic coefficients, KA and KD, were determined using the BiaEvaluation software (GE Healthcare, Diegem, Belgium) and best fit was determined with a 1 : 1 binding model with drifting baseline and local Rmax. All experiments were repeated in triplicate on at least three occasions.
Nta chip
Manufactured by GE Healthcare
Sourced in Belgium
The NTA chip is a laboratory equipment product designed for affinity-based protein purification. It is a solid-state support matrix that utilizes nitrilotriacetic acid (NTA) to bind and purify histidine-tagged proteins. The NTA chip provides a convenient and efficient platform for the capture, purification, and isolation of target proteins from complex biological samples.
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Lab products found in correlation
Biacore x100 system, by GE Healthcare (1 mentions)
Hrp conjugated goat anti mouse igg, by Jackson ImmunoResearch (1 mentions)
Hoechst 33342 trihydrochloride, by Thermo Fisher Scientific (1 mentions)
Streptavidin magnesphere paramagnetic particles, by Promega (1 mentions)
Alexa fluor 488 conjugated goat anti human igg, by Jackson ImmunoResearch (1 mentions)
Hitrap mabselect sure 5 ml column, by GE Healthcare (1 mentions)
Mouse monoclonal anti flag m2 antibody, by Merck Group (1 mentions)
Sypro orange, by Thermo Fisher Scientific (1 mentions)
Prolong gold antifade reagent, by Thermo Fisher Scientific (1 mentions)
Biacore 100, by GE Healthcare (1 mentions)
Hbs p buffer, by GE Healthcare (1 mentions)
Biaevaluation software, by GE Healthcare (1 mentions)
Biacore 3000, by GE Healthcare (1 mentions)
Surfactant p20, by GE Healthcare (1 mentions)
Biacore s200, by GE Healthcare (1 mentions)
9 protocols using nta chip
1
Quantifying His-ClfA1–520-vWbp Interaction
2
SPR Analysis of CesT-CsrA Interactions
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The SPR analyses were carried out using BIAcore X-100 system with the NTA chips (GE Healthcare) at 25 °C. For the SPR measurements, all the proteins, including CesT, CsrA, and Re-CsrA, were prepared in a PBST buffer consisting of 1× PBS (136 mM NaCl, 2.6 mM KCl, 8 mM Na2HPO4, 2 mM KH2PO4, pH 7.4) and 0.005% Tween 20. The CesT protein was immobilized on the Ni-NTA chip at ~50 response units. The blank channel served as the negative control. When the data collection was finished in each cycle, the sensor surface was regenerated with 350 mM EDTA and then with 0.5 mM NiCl2. Gradient concentrations of CsrA or Re-CsrA ranging from 16 nM to 256 nM were flowed over CesT on the chip-surface at 30 μl/min, and the amount of bound proteins in response units was recorded for comparison.
The kinetic analyses were analyzed with BIAevaluation software 4.1. For dissociation constant (KD) calculations, the data of CsrA binding to CesT were fitted to a Bivalent-Analyte binding model, whereas the data of Re-CsrA binding to CesT were fitted to a Steady-State Affinity model.
The kinetic analyses were analyzed with BIAevaluation software 4.1. For dissociation constant (KD) calculations, the data of CsrA binding to CesT were fitted to a Bivalent-Analyte binding model, whereas the data of Re-CsrA binding to CesT were fitted to a Steady-State Affinity model.
3
Binding Affinity Analysis of JN241 Mutants
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In each cycle of binding test between JN241 mutants with APJ nanodisc, the NTA chips (28994951, GE Healthcare) were preconditioned with 1-min pulses of 350 mM EDTA at pH 8.3. NiCl2 (500 μM) was then injected for 90 s. His-tagged APJ nanodiscs were captured at a testing surface around 400 response unit (RU) captured level for testing. Four different doses (12.5, 25, 50, and 100 nM) of JN241 mutant antibodies were sequentially injected into both control and testing flow chambers. The binding curve (resonance unit against time) was obtained after deduction of the signaling from control surface and shown in the sensorgram. After antibody injection, 10 mM glycine-HCl (pH 1.5) and 350 mM EDTA were sequentially injected to regenerate the surface for the next round of testing.
4
Comprehensive Molecular Biology Protocol
5
SPR Kinetics of Keap1-Compound Interactions
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SPR of Keap1 was performed in HBSN buffer with 0.005% P20, 1% DMSO, and 0.5 mM TCEP. An NTA chip (GE Healthcare) was conditioned with 0.5 M EDTA (60 s) followed by 10 mM NaOH (30 s). The NTA surface was pre-equilibrated with 0.5 mM NiCl2 (15 s) and Keap1 was captured by injecting a 100 nM solution of murine Keap1(322–624)C-10His (30 s). Compound titrations were run using a 5 point 3-fold dilution single cycle kinetics method with a top concentration of 100 nM. Association and dissociation times were set to 60 and 500 s respectively. Prior to titrating compounds, a series of buffer injections were carried out to serve as blanks. The flowrates for conditioning, Keap1 capture, and compound titrations were 30, 10, and 50 uL/min respectively. To ensure an un-complexed target at the start of each titration, following each compound titration, Keap1 was removed from the surface using EDTA, the surface was pre-treated with NiCl2, and Keap1 was re-captured as described above.
6
Immobilization of Ion Channel Domains
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The purified domains of KCNH and HCN channels were immobilized on a NTA chip (GE Healthcare), as previously described [34 (link)]. Immobilizations of the proteins were performed in HBS-P buffer (150 mM NaCl, 10 mM HEPES, 0.05% (v/v) surfactant P20, pH 7.4). First the NTA sensor surface was activated with a 1 min injection of 0.5 mM NiCl2. The coupling of the Ni2+-NTA chip surface groups with the 6-His-tagged proteins was then achieved by 2.5 min injections of the proteins at ~ 200 nM concentrations for high density (HD) and ~ 10 nM for low density (LD) immobilization on the chip surface. After the initial capturing, the proteins were covalently cross-linked via 20 s injections of NHS-EDC carboxyl-reactive cross-linkers to prevent protein loss from the chip surface with successive analyte and buffer injections. This was followed by 20 s injection of 1 M ethanolamine to block the remaining reactive sites. The proteins were captured at ~ 1000–3000 RU (1 RU = 1 pg of protein per mm2).
7
Kinetic Binding Assay of HIS-CREB
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BIACORE 100 (GE Healthcare) was employed to perform surface plasmon resonance. Briefly, purified HIS-CREB proteins were captured in the test channel of an NTA-chip (GE, Healthcare) in HEPES buffer (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, pH 7.5). APC solutions with different concentrations were flowed through immobilized HIS-CREB proteins for indicated times, which caused equilibrium response values (RU) to change appropriately. The detected RU values were plotted by time using GraphPad Prism software.
8
SPR Analysis of ERK2 Binding Kinetics
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All SPR experiments were carried out on a BIAcore S200 (GE Healthcare) at 25 °C. ERK2 protein with an N-terminal 12His-tag was immobilised on an NTA chip (GE Healthcare) using the capture-coupling method as described by the manufacturer to achieve an immobilisation level of 3000–4000 resonance units (RU). The immobilisation buffer was 50 mM TRIS/HCl, 300 mM NaCl, 5 mM MgCl2, 2 mM TCEP, 0.05% (v/v) surfactant P20 at pH 8. Compound binding measurements were performed at a flow rate of 50 μL min–1 in the same running buffer used for the immobilisation supplemented with 2% DMSO. GDE-TCO-2 was tested in a in a multi-cycle format (concentration series from 5000 nM, as eight point three-fold serial dilutions). Each concentration was injected for 240 s and the dissociation was monitored for 900 s. The binding kinetics for SCH-TCO were measured in a single-cycle format and the dissociation was monitored for 3600 s. Five concentrations of SCH-TCO, prepared in three-fold serial dilution from 50 nM, were injected for 240 s. Solvent corrected, and background subtracted binding curves were analyzed using the Biacore insight evaluation software following standard procedures and data were fitted to a 1 : 1 binding model.
9
PfAtg8 Protein Binding Kinetics
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His12-PfAtg8CM was
injected over an NTA-chip (GE Healthcare) preconditioned with
nickel, leading to capture of 3000 response units (RUs). Running buffer
contained 3% DMSO. A 2-fold-dilution series of compounds, highest
concentration of 75 μM, was injected over PfAtg8 variants at 40 μL/min.
injected over an NTA-chip (GE Healthcare) preconditioned with
nickel, leading to capture of 3000 response units (RUs). Running buffer
contained 3% DMSO. A 2-fold-dilution series of compounds, highest
concentration of 75 μM, was injected over PfAtg8 variants at 40 μL/min.
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