SPR was performed using a Biacore T200 (Cytiva) and Series S Sensorchip SA (Cytiva, BR100531) at the temperature of 25 °C. A solution of PBS, 0.005% TWEEN20 (Sigma, P9416), 0.5 mM TCEP (Nacalai Tesque, 06342-21), and 2% DMSO (Sigma, 276855) was filtered with a 0.22-μm filter and used as the running buffer. Kinetic measurements were carried out at a flow rate of 30 μL/min, and the association and dissociation phases were monitored for 120 s and 120–300 s, respectively. The G9a SET domain was biotinylated by using NHS-PEG4-Biotin (Thermo, 39259), and captured to flow cells 2, 3, and 4 with immobilization levels of ~2300 RU. No specific treatment was applied to the reference surface (flow cell 1). To prevent oxidation of the immobilized protein, a solution containing 1 μM ZnCl2 and 5 mM TCEP was injected at a flow rate of 10 μL/min for 60 s before each set of kinetic measurements. The extra wash command was executed after each measurement cycle with 50% DMSO aqueous solution. To correct for bulk responses, solvent correction was performed once after all kinetics runs by using running buffer containing 1–3% DMSO. The sensorgrams obtained from the three flow cells were analyzed individually by Biacore T200 Evaluation Software (Cytiva). The dissociation constant (KD) and binding kinetic parameters were calculated by a curve fit to a built-in 1:1 binding model.
Series s sensor chip sa
Manufactured by Cytiva
Sourced in United States, New Zealand
The Series S Sensor Chip SA is a lab equipment product designed for surface plasmon resonance (SPR) analysis. It provides a gold-coated sensor surface for immobilizing various biomolecules, enabling the study of molecular interactions in real-time.
Automatically generated - may contain errors
Lab products found in correlation
Biacore t200, by Cytiva (2 mentions)
Nhs peg4 biotin, by Thermo Fisher Scientific (1 mentions)
Biacore t200 evaluation software, by Cytiva (1 mentions)
Tween 20, by Merck Group (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Ez link nhs biotin reagent, by Thermo Fisher Scientific (1 mentions)
Biacore s200, by Cytiva (1 mentions)
Biacore t100 evaluation software, by Cytiva (1 mentions)
Biacore t100, by Cytiva (1 mentions)
Biacore 4000, by Cytiva (1 mentions)
6 protocols using series s sensor chip sa
1
Kinetic Analysis of G9a SET Domain
2
Competitive Inhibition of RBD-hACE2 Interaction
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The interaction between RBD and hACE2 was competitively inhibited by the mice serum. The competition assay was determined by surface plasmon resonance (SPR), using a Biacore S200 instrument (Cytiva Life Sciences, USA). RBD was biotinylated by the EZ-link-NHS-biotin reagent (Thermo Fisher Scientific). The biotinylated RBD (5 μg/mL) was injected over the Series S Sensor chip SA (Cytiva Life Sciences, USA) for 30 s at 10 μL/min. The sera of the RBD-IC28-M group on day 21 were harvested. An aliquot of serum from each mouse (5 μL) was mixed to obtain a mix representative of the RBD-IC28-M group. A two-fold dilution of the sera was loaded on the SA Sensor chip to saturate RBD for 300 s. Then, 400 nM hACE2 was associated with the chip for 300 s to detect the competitive binding signal. The chips were then regenerated with 10 mM glycine-HCl buffer (pH 2.5). Real-time signal data were collected, and the competition behavior was recorded.
3
Surface Plasmon Resonance Binding Assay
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Binding studies were conducted on a Biacore T100 (Cytiva). SPR data were collected using Biacore T100 control software version 2.0.4. Approximately, 300 response units (RU) of site-specifically biotinylated IL-17RA, IL-17RB or unrelated negative-control reference protein were immobilized onto a Series S Sensor Chip SA (Cytiva). For each binding cycle, a regeneration condition of 2 M MgCl2 was used. Data were processed using Biacore T100 Evaluation Software, version 2.0.4 (Cytiva). Dissociation constants were calculated using steady-state affinity analysis.
4
Kinetic Analysis of Peptide-Protein Interactions
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Using a Biacore T200 (Cytiva), we captured the biotin-labeled peptides on the surface of a Series S Sensor Chip SA (Cytiva). We serially diluted purified His-tagged CNA in HBS-EP + (10 mM HEPES, pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.05% surfactant P20) and passed it sequentially over three flow cells (flow cell 1: biotin-VEET, flow cell 2: biotin-CABIN1-L-1, flow cell 3: biotin-p-CABIN1-L-1). Each cycle was performed at 25 °C at a flow rate of 30 μl/min with 120 s of contact and 240 s of dissociation. We analyzed the kinetics of interactions using BIAevaluation 3.2 RC1 (Cytiva).
5
Surface Plasmon Resonance Binding Kinetics
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The surface plasmon resonance
(SPR) experiments were carried out using a Biacore 8K optical biosensor
and Series S sensor Chip SA (Cytiva, Marlborough, MA). The chips were
washed three times with 1 M NaCl/50 mM NaOH. Proteins were injected
at a flow rate of 5 μL/min in SPR immobilization buffer (50
mM HEPES, 150 mM NaCl, 0.05% (v/v) Tween 20, 0.25 mM TCEP, and 0.05%
(w/v) BSA, pH 7.5) for 875 s. The experiments were performed at 278
K with a flow rate of 50 μL/min in an SPR running buffer (SPR
immobilization buffer containing 1% (v/v) DMSO). The tested analytes
were diluted in an SPR running buffer. After baseline equilibration
with a series of buffer blanks, a DMSO correction was performed from
1 to 3%. Eight analyte concentrations were injected sequentially (160
s), with a short dissociation between each injection. The last injection
was followed by a 2000 s dissociation step. The data were fitted with
the Single-Cycle Kinetic module of Biacore 8K evaluation software
using a 1:1 binding model.
(SPR) experiments were carried out using a Biacore 8K optical biosensor
and Series S sensor Chip SA (Cytiva, Marlborough, MA). The chips were
washed three times with 1 M NaCl/50 mM NaOH. Proteins were injected
at a flow rate of 5 μL/min in SPR immobilization buffer (50
mM HEPES, 150 mM NaCl, 0.05% (v/v) Tween 20, 0.25 mM TCEP, and 0.05%
(w/v) BSA, pH 7.5) for 875 s. The experiments were performed at 278
K with a flow rate of 50 μL/min in an SPR running buffer (SPR
immobilization buffer containing 1% (v/v) DMSO). The tested analytes
were diluted in an SPR running buffer. After baseline equilibration
with a series of buffer blanks, a DMSO correction was performed from
1 to 3%. Eight analyte concentrations were injected sequentially (160
s), with a short dissociation between each injection. The last injection
was followed by a 2000 s dissociation step. The data were fitted with
the Single-Cycle Kinetic module of Biacore 8K evaluation software
using a 1:1 binding model.
6
Measuring SARS-CoV-2 Spike Protein Binding
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VHH affinities and VHH-Fc bivalent apparent affinities were measured on a Biacore 4000 (Cytiva). SARS-CoV-2 PreS Spike ECD protein was biotinylated via a C-terminal Avi tag and immobilized at 600 RU to 1500 RU on a streptavidin coated Series S Sensor Chip SA (Cytiva, Cat. No. 29699621). VHHs were injected at 3.7 nM to 300 nM and VHH-Fcs were injected at 2.5 nM to 200 nM. Injections were 3 min long and dissociation was monitored for 10 min. The running buffer was HBS EP + (10 mM HEPES, 150 mM NaCl, 0.05% Tween20, pH 7.4) and 25 °C. The surface was regenerated with a 30 s injection of Glycine pH 1.9. Data were fit to a 1:1 binding model using Biacore Evaluation software version 1.1.
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