The SPR analyses were carried out using the Biacore 8K surface plasmon resonance instrument (Cytiva, Marlborough, MA, USA). A total of 100 nM His6-14-3-3ε was immobilized on the NiCl2-activated NTA chip (Cytiva, Marlborough, MA, USA) as previously described [34 (link)]. Then, different concentrations of peptide in the range of 0−500 μM (depending on the peptide) were injected. The experiments were performed in 20 mM TRIS, 150 mM NaCl, 50 μM EDTA, 0.005% Tween 20, and pH 7.4 buffer. The affinities of the peptides binding to 14-3-3ε were determined by fitting the data to a steady-state binding model using Biacore Insight Evaluation Software (version 5.0.18.22102).
Nicl2 activated nta chip
Manufactured by Cytiva
Sourced in United States
The NiCl2-activated NTA chip is a lab equipment product designed for protein purification. It utilizes nitrilotriacetic acid (NTA) chemistry to immobilize proteins of interest. The chip is pre-activated with nickel ions (NiCl2) to enable the capture of His-tagged proteins. This product provides a simple and efficient method for affinity-based protein purification in research and analytical applications.
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Lab products found in correlation
2 protocols using nicl2 activated nta chip
1
Binding Affinity of Peptides to 14-3-3ε
2
Measuring Binding Affinities of pT Peptide Analogs for 14-3-3ε
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Binding
affinities of pT peptide analogs for 14-3-3ε were measured by
SPR using a Biacore 8K Surface plasmon resonance instrument (Cytiva,
Marlborough MA, USA). 100 nM 14-3-3ε-(His)12 protein
in 20 mM Tris, 150 mM NaCl, 50 μM EDTA, 0.005% Tween 20, pH
7.4 buffer was immobilized to a NiCl2 activated NTA chip
(Cytiva, Marlborough MA, USA) at a flow rate of 5 μL/min for
10 min and then washed with the running buffer for 20 min. Then peptide
solutions at a concentration range of 1 nM–2000 nM were injected
cycle-wise at the flow rate of 30 μL/min for 1 min, and dissociation
time was set for 3 min. All SPR experiments were performed at 25 °C.
The affinities and kinetics of the peptides binding to 14-3-3ε
were determined by steady-state and 1:1 binding models, respectively,
using Biacore Insight Evaluation Software (version 5.0.18.22102).
At least three replicates were done for each peptide at similar conditions,
and binding affinities (KD) are presented
as mean ± SD. KD values were used
to calculate binding free energy (ΔG) with
the following formula: ΔG = RT ln KD (R = 1.986 cal mol–1 K–1, T = 298 K).
affinities of pT peptide analogs for 14-3-3ε were measured by
SPR using a Biacore 8K Surface plasmon resonance instrument (Cytiva,
Marlborough MA, USA). 100 nM 14-3-3ε-(His)12 protein
in 20 mM Tris, 150 mM NaCl, 50 μM EDTA, 0.005% Tween 20, pH
7.4 buffer was immobilized to a NiCl2 activated NTA chip
(Cytiva, Marlborough MA, USA) at a flow rate of 5 μL/min for
10 min and then washed with the running buffer for 20 min. Then peptide
solutions at a concentration range of 1 nM–2000 nM were injected
cycle-wise at the flow rate of 30 μL/min for 1 min, and dissociation
time was set for 3 min. All SPR experiments were performed at 25 °C.
The affinities and kinetics of the peptides binding to 14-3-3ε
were determined by steady-state and 1:1 binding models, respectively,
using Biacore Insight Evaluation Software (version 5.0.18.22102).
At least three replicates were done for each peptide at similar conditions,
and binding affinities (KD) are presented
as mean ± SD. KD values were used
to calculate binding free energy (ΔG) with
the following formula: ΔG = RT ln KD (R = 1.986 cal mol–1 K–1, T = 298 K).
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