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).
Biacore 8k surface plasmon resonance instrument
Manufactured by Cytiva
Sourced in United States
The Biacore 8K is a surface plasmon resonance (SPR) instrument designed for real-time, label-free analysis of biomolecular interactions. It enables the study of affinity, kinetics, and specificity of a wide range of biomolecular interactions, including those between proteins, peptides, nucleic acids, small molecules, and cells.
Automatically generated - may contain errors
Lab products found in correlation
2 protocols using biacore 8k surface plasmon resonance instrument
1
Binding Affinity of Peptides to 14-3-3ε
2
Measuring Binding Affinities of pT Peptide Analogs for 14-3-3ε
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
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).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!