Binding analyses involving NRP1 and its partners, either Cc or histones, were performed by Surface Plasmon Resonance (SPR) using CO SPRi-Biochips and a SPRi-Plex II (Horiba). Cc, H2B, H3 and H4 histones, as well as H2A–H2B and H3–H4 histone dimers, were covalently attached to the matrix using CO direct amine-coupling chemistry. Proteins were immobilized at 1, 5 and 10 μM for Cc, H2B, H3 and H4 histones, whereas for the H2A–H2B and H3–H4 histone dimers concentrations were 0.1, 1 and 10 μM. Bovine serum albumin was attached to the chip surface at reference spots used as control. The binding measurements were performed at 25°C in 10 mM sodium phosphate buffer, pH 7.4. NRP1 at various concentrations (from 0.1 to 1 μM) were flowed on the surface at a 50 μl/min rate. Each solution was injected three times at least. The signals from the reference spot surface were subtracted in every sensogram. The data were analysed with Origin 2016 (OriginLab Corporation).
Spri plex 2
Manufactured by Horiba
Sourced in United States, France
The SPRi-Plex II is a surface plasmon resonance imaging (SPRi) system designed for label-free, real-time biomolecular interaction analysis. It utilizes the principle of surface plasmon resonance to detect and monitor changes in the refractive index at the sensor surface, allowing for the study of a wide range of biomolecular interactions without the need for labeling.
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Lab products found in correlation
7 protocols using spri plex 2
1
NRP1 Binding Analyses by SPR
2
SPRi Characterization of SET/TAF-Iβ ΔC
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Surface plasmon resonance imaging (SPRi) experiments were carried out using a SPRi-Plex II (Horiba) equipment. SET/TAF-Iβ ΔC species at different concentrations (5 and 10 μM) were covalently coupled to a CO-chemistry biochip surface. Bovine serum albumin (BSA) was coupled at reference spots as a control. Cc solutions at 500 nM, 2.5 μM and 5 μM were passed, at least three times each, onto the biochip surface at a 50 μL/min flow rate. Measurements were performed at 25 °C, using 10 mM sodium phosphate (pH 6.3) as running buffer. Data processing and analysis was performed using Origin 2016 (OriginLab Corporation). SPR response curves were obtained as average of the three independent injections and simultaneously fitted to a one-site binding model.
3
SPRi-based Interactome Profiling of Icaritin
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The SPRi measurements were performed in a temperature‐controlled HORIBA Scientific SPRi‐PLEX II apparatus. Briefly, Icaritin was added onto the photo‐crosslingker chip and was irradiated with UV photocrosslinker chamber at 365 nm wavelength for 15 min. After washed with enthanol and water, protein lysate from 1 × 107 SMMC‐7721 cells was loaded onto the chip and washed with PBS to remove unbound proteins. Then the bound proteins were eluted with 1% SDS and reloaded onto the chip for a second round screening. After two rounds screening, the eluted samples were subject to LC‐MS profiling.
4
Surface Plasmon Resonance Imaging
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The SPRi measurements were performed in a temperature-controlled HORIBA Scientific SPRi-PLEX II apparatus. The sensing mechanism utilized an incident monochromatic light that hits the sensor surface, a detector that collects the resulting reflected light and a CCD camera that generates a real-time contrast image. The sensor surface consists of a high index prism, coated with and a gold layer (50 nm) where the ligands of interest are immobilized. At a specific incident angle known as the resonance angle, the impinging light excites the free electrons in the gold layer; resulting in an evanescent field (up to ~400 nm); which exponentially decays out from the surface. And this in turn leads to a dip in the reflected light. The surface plasmons thus created are sensitive to perturbations in the refractive index of the surrounding medium; and a detector measures these alterations as changes in the reflectivity signal. The SPRi apparatus also allows a real-time visualization of the sensing surface for multi-array purposes by intercepting the reflected light with a CCD camera to provide a contrast image reflecting biomolecular interactions at the surface.
5
SPR Imaging of Host-Guest Polymer Interactions
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First the SPRi system was filled at high flow rate (500 μL/min) with the running buffer (ultrapure water).
Then the spotted SPRi-Biochip™ was inserted into the analysis chamber of the SPRi-Plex II™ instrument (HORIBA Scientific). The flow rate was fixed at 50 μL/min and the experiment was carried out at 25 °C.
Each region of interest (HP spots and water spots) was selected and identified.
The SPRi system allows plotting plasmon curves (the reflectivity variation versus incidence angle) for each spot in order to monitor the reflectivity variation at a fixed incidence angle chosen where the plasmon slope is globally higher for each spot.
Two solutions of the guest polymer (GP) diluted in ultrapure water at 1.0 g/L and 0.1 g/L were each sequentially injected in the flow cell for 5 min. Between each GP injection, the chip was rinsed with ultrapure water to remove unbound guest polymer for around 15 min.
The interaction between the two polymers (host and guest) was monitored in real time by SPR imaging. The reflectivity variations of the chip active spotted matrix and hence the host polymer/guest polymer interactions were measured simultaneously for the complete chip matrix.
6
SPRi Kinetic Analysis of Phosphatase Binding
7
Multiplexed SPR Analysis of Foldamer Interactions
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A HORIBA SPRi-Plex II (HORIBA Jobin Yvon S.A.S. Palaiseau, France) surface plasmon resonance imaging (SPRi) system was used for multiplexed SPR measurements at a fixed working angle selected for optimal response in the PBS running buffer (10 mM phosphate, 137 mM NaCl, 2.7 mM KCl). The sensitivity across the active area of the chip was normalized using the refractive index dependent signal change for 180 mM NaCl containing PBS.
The foldamer interactions were measured in PBS running buffer at a flow rate of 50 µL min -1 and 25.00 °C. The signal change recorded for control (Figure S7) was used as a negative control and subtracted from the signals recorded in the various foldamer spots. The injected volume for each analyte and regeneration solution was 180 µL. 5 mM NaOH solution was used (3.6 min, flow rate 50 µL min -1 ) to regenerate the foldamer-modified surfaces after each interaction. Kinetic evaluation of binding interactions was performed with global analysis using first order kinetics with Scrubber 2 (GenOptics version, BiaLogic Sofware, Campbell, Australia). Detailed procedure for spotting SPR sensor chips is found in the supporting text.
The foldamer interactions were measured in PBS running buffer at a flow rate of 50 µL min -1 and 25.00 °C. The signal change recorded for control (Figure S7) was used as a negative control and subtracted from the signals recorded in the various foldamer spots. The injected volume for each analyte and regeneration solution was 180 µL. 5 mM NaOH solution was used (3.6 min, flow rate 50 µL min -1 ) to regenerate the foldamer-modified surfaces after each interaction. Kinetic evaluation of binding interactions was performed with global analysis using first order kinetics with Scrubber 2 (GenOptics version, BiaLogic Sofware, Campbell, Australia). Detailed procedure for spotting SPR sensor chips is found in the supporting text.
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