To evaluate the target-binding of the selected ZEBVLMP-2 affibodies to EBV LMP-2, surface plasmon resonance (SPR) was performed on a ProteOn XPR36 system (Bio-rad, California, USA). The LMP-2 B-epitope fusion protein (1 nM) served as the ligand was immobilized onto the surface of carboxylate glucans in HTG sensor chip (Bio-rad), as described previously [30 (link),31 (link)]. Subsequently, five or six concentrations of each affibody sample were prepared and injected over the chip surface to record sample binding to the surface. All experiments were carried out with a flow rate of 30 μL/min at 25°C. SPR data sets were fit globally using a 1:1 Langmuir binding model and analyzed by BIA evaluation 3.0.2 software.
Htg sensor chip
Manufactured by Bio-Rad
The HTG sensor chip is a laboratory equipment product designed for use in various analytical procedures. Its core function is to serve as a platform for the detection and measurement of specific analytes or targets. The sensor chip provides the necessary infrastructure to facilitate these analytical processes, though its specific intended uses are not detailed here.
Automatically generated - may contain errors
4 protocols using htg sensor chip
1
Evaluating Z_EBVLMP-2 Affibody Binding to EBV LMP-2
2
Quantifying PCNA-Antibody Interactions
3
Affibody Binding Kinetics to MOMP
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To evaluate the target binding of the selected ZMOMP affibodies to MOMP, surface plasmon resonance (SPR) was performed on a ProteOn XPR36 system (Bio-rad, CA, USA). The MOMP fusion protein (1 nM) served as the ligand was immobilized onto the surface of carboxylate glucans in HTG sensor chip (Bio-rad) and PBS was used as running buffer and for dilution of the analytes, as described previously (Zhu et al. 2020b (link); Xue et al.2016; Zhu et al. 2018 (link)). Subsequently, five or six concentrations of each affibody sample ranging from 125 to 4000 nM were prepared and injected over the chip surface to record sample binding to the surface. ZWT affibody was set as a negative control. All experiments were carried out with a flow rate of 30 μL/min at 25 °C. The kinetic constants, including the association constant (ka), dissociation constant (kd), and affinity (KD, KD = kd/ka), were calculated by BIAcore T200 evaluation 3.0.2 software provided by the manufacturer, according to a 1:1 Langmuir binding model.
4
SPR Analysis of LC8 Binding Peptides
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The full-length His6-tagged LC8 (DYNLL2; UniProt accession number Q96FJ2) was cloned into a pET21-derived pBH4 vector and expressed as described previously [84 (link)]. The 11-residue-long fragments of the predicted binding partners were synthesized using solid-phase technique by GenoSphere Ltd.
SPR measurements were performed on a ProteOn XPR36 (Bio-Rad) instrument equipped with HTG sensor chip (Bio-Rad). The sensor chip was activated for 300 s with 150 μl 10 mM NiCl2 solution followed by a washing step for 300 s with the running buffer containing 20 mM Hepes, 150 mM NaCl, 0.05% Tween-20, 0.1 mM TCEP, 50 μM EDTA, pH 7.5 buffer. The immobilization of His6-tagged LC8 was performed on the activated Ni2+-NTA surface at three different densities (1200 RU, 900 RU, 600 RU). The putative binding peptides were injected onto the chip at five different concentrations simultaneously at a flow rate of 60 μl ∙ min-1 for 400 s, while the dissociation of the peptides was recorded for 600 s. In the sixth analyte channel, running buffer was injected for double referencing. The double referenced data were global fitted to the 1: 1 Langmuir model using the ProteOn software. The presented Kd values were obtained from the mean of the kinetic and the equilibrium analysis delivered Kd values, and the standard deviations were calculated from three individual Kd values.
SPR measurements were performed on a ProteOn XPR36 (Bio-Rad) instrument equipped with HTG sensor chip (Bio-Rad). The sensor chip was activated for 300 s with 150 μl 10 mM NiCl2 solution followed by a washing step for 300 s with the running buffer containing 20 mM Hepes, 150 mM NaCl, 0.05% Tween-20, 0.1 mM TCEP, 50 μM EDTA, pH 7.5 buffer. The immobilization of His6-tagged LC8 was performed on the activated Ni2+-NTA surface at three different densities (1200 RU, 900 RU, 600 RU). The putative binding peptides were injected onto the chip at five different concentrations simultaneously at a flow rate of 60 μl ∙ min-1 for 400 s, while the dissociation of the peptides was recorded for 600 s. In the sixth analyte channel, running buffer was injected for double referencing. The double referenced data were global fitted to the 1: 1 Langmuir model using the ProteOn software. The presented Kd values were obtained from the mean of the kinetic and the equilibrium analysis delivered Kd values, and the standard deviations were calculated from three individual Kd values.
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