Biacore cm5 sensor chip

Manufactured by GE Healthcare

Sourced in Sweden, United Kingdom

The Biacore CM5 sensor chip is a lab equipment product designed for use with Biacore systems. The chip serves as a surface for the immobilization of molecules, allowing for real-time analysis of biomolecular interactions.

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25 protocols using biacore cm5 sensor chip

Kinetics of Aβ42 Protofibrils Binding

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Surface plasmon resonance (SPR) studies were performed on a Biacore X100 instrument (GE Healthcare). The Aβ₄₂cc protofibrils were immobilized onto a Biacore CM5-sensor chip (GE Healthcare), as described previously^{31 (link)}.
Five or six concentrations of each analyte were prepared in HBS-EP (10 mM HEPES, 150 mM NaCl, 3 mM ETDA, 0.005% Tween-20, pH 7.4) and injected over the immobilized chip surface for 250 s to record analyte binding to the surface. Dissociation was observed for 2,000 s in running buffer. The sensor surface was regenerated after each injection with 20 mM NaOH with 90 s contact times. All experiments were carried out at 25 °C with a flow rate of 10 μL/min.
SPR data sets were analyzed using Biacore X100 Evaluation 2.0.1 software and curve fitting was performed with a heterogeneous binding site model using global kinetic fitting, but with local adjustment of the parameter R_max.

Wahlberg E., Rahman M.M., Lindberg H., Gunneriusson E., Schmuck B., Lendel C., Sandgren M., Löfblom J., Ståhl S, & Härd T. (2017). Identification of proteins that specifically recognize and bind protofibrillar aggregates of amyloid-β. Scientific Reports, 7, 5949.

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Biacore T200 SPR Analysis of Eno1

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Biacore T200 instrument (GE Healthcare) combined with a CM5 sensor chip (GE Healthcare) was used for SPR analysis. C. albicans Eno1 protein was immobilized in parallel-flow channel on a BIAcore™ CM5 sensor chip using the Amine Coupling Kit (GE Healthcare). Serial dilutions of mAb 12D9 were injected into the flow system. Experiments were conducted using PBS as running buffer, and the analyte was injected at a flow rate of 30 μl/min. The association time was 90 s and the dissociation time was 60 s. The affinity constants for binding were obtained using BIA evaluation software and a 1:1 Langmuir binding model.

Chen S.M., Zou Z., Guo S.Y., Hou W.T., Qiu X.R., Zhang Y., Song L.J., Hu X.Y., Jiang Y.Y., Shen H, & An M.M. (2020). Preventing Candida albicans from subverting host plasminogen for invasive infection treatment. Emerging Microbes & Infections, 9(1), 2417-2432.

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Measuring FH Variant Decay Kinetics

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To estimate the ability of each FH variant to accelerate the rate at which the C3 convertase (C3bBb, i.e., a binary complex of C3b and Bb) decays, we used a well-established SPR-based assay²⁹ (link) performed at 25°C on a Biacore T200 instrument. In this technique, the dissociation of Bb from surface-immobilized C3bBb is monitored as a function of time. Thus, 2845 RUs of C3b (Complement Technology) were attached, using standard amine coupling, to a Biacore CM5 sensor chip (GE Healthcare), after which C3bBb was assembled on the chip surface as previously described.²⁸ (link) Its decay was subsequently monitored in the presence or absence of injected FH. Data were analyzed using the Biacore Evaluation software. See Supplementary Methods III for details.

Biggs R.M., Makou E., Lauder S., Herbert A.P., Barlow P.N, & Katti S.K. (2022). An Evaluation of the Complement-Regulating Activities of Human Complement Factor H (FH) Variants Associated With Age-Related Macular Degeneration. Investigative Ophthalmology & Visual Science, 63(12), 30.

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Biacore Binding Assay for Nef Protein Interactions

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The measurements were carried out with a Biacore T100 instrument (GE Healthcare). A Biacore CM5 Sensor Chip and an amine coupling kit were purchased directly from GE Healthcare. The suitable pH value of 4.5 for His-Nef or His-Nef ^E63A/F68A immobilization (20 μg/ml in 10 mM acetate buffer) was determined first. The CM5 censor chip was activated and then injected with His-Nef and His-Nef ^E63A/F68A (20 μg/ml, in 10 mM acetate buffer, pH 4.5) for 7 min into channel 2 and 4, respectively. The residual activated groups on the surfaces were blocked with an injection of ethanolamine HCl (1 M) for 7 min. The lovastatin or fluvastatin was diluted at the indicated range of concentrations. Binding to the His-Nef or His-Nef ^E63A/F68A protein was monitored for about 60 s. The dissociate time was 120 s for lovastatin or fluvastatin with running buffer in per cycle.

Liu B., Zhang X., Zhang W., Wu L., Jing S., Liu W., Xia B., Zou F., Lu L., Ma X., He D., Hu Q., Zhang Y., Deng K., Cai W., Tang X., Peng T., Zhang H, & Li L. (2019). Lovastatin Inhibits HIV-1-Induced MHC-I Downregulation by Targeting Nef–AP-1 Complex Formation: A New Strategy to Boost Immune Eradication of HIV-1 Infected Cells. Frontiers in Immunology, 10, 2151.

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SPR Analysis of PfLysRS-ASP3026 Interaction

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PfLysRS was chemically immobilized on a Biacore CM5 sensorchip (immobilization level ∼10691 resonance unit, RU) at pH 5.5 according to the immobilization kit (GE Healthcare). PfLysRS (33 nM) was captured for 90 s at a flow of 30 μl·min⁻¹. Affinity between PfLysRS and ASP3026 was measured by SPR on a Biacore T200 (GE Healthcare) at 25°C with a running buffer (0.02 M phosphate buffer pH 7.4, 2.7 mM KCl, 0.137 M NaCl, 1 mM l-lysine, and 0.05% Surfactant P20). ASP3026 dilutions were made to yield (dissolved in running buffer) at concentrations of 1000, 500, 250, 125 and 62.5 nM (association time: 90 s, dissociation time: 120 s, flow: 30 μl·min⁻¹). To study the ATP competitive nature of ASP3026, 1 mM ATP or 4 μM cladosporin analog 3-(cyclohexylmethyl)-6,8-dihydroxy-1H-isochromen-1-one was included in the running buffer. Kinetic evaluation of the interaction between LysRS and ASP3026 was performed by global fitting of the data to a 1:1 interaction model using Biacore Evaluation Software 3.1 (GE Healthcare).

Zhou J., Huang Z., Zheng L., Hei Z., Wang Z., Yu B., Jiang L., Wang J, & Fang P. (2020). Inhibition of Plasmodium falciparum Lysyl-tRNA synthetase via an anaplastic lymphoma kinase inhibitor. Nucleic Acids Research, 48(20), 11566-11576.

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Binding Kinetics of Viral Proteins

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The measurements of Ferritin, RBD monomers, RBD-Ferritin nanoparticles and RBD-HR_Ferritin nanoparticles binding to hACE2 were carried out with a BIAcore T100 instrument (GE Healthcare). A BIAcore CM5 Sensor Chip and an amine coupling kit were purchased from GE Healthcare. The suitable pH value of 4.5 for hACE2 immobilization was determined. The CM5 censor chip was activated and then injected with hACE2 (2 μg/mL, in 10 mM acetate buffer, pH 4.5) for 7 min. The residual activated groups on the surfaces were blocked with an injection of ethanolamine HCl (1 M) for 7 min. Ferritin only nanoparticles, RBD only monomers, RBD-Ferritin nanoparticles and RBD-HR-Ferritin nanoparticles were diluted into different concentrations and then injected (30 μl/min). hACE2-bound protein was monitored for about 120 s for each protein. The dissociation time was 200 s with running buffer per cycle. The association rate (‘on rate’, Ka) and dissociation rate (‘off rate’, Kd) were measured, followed by the calculating of equilibrium dissociation constant (‘binding constant’, KD).

Ma X., Zou F., Yu F., Li R., Yuan Y., Zhang Y., Zhang X., Deng J., Chen T., Song Z., Qiao Y., Zhan Y., Liu J., Zhang J., Zhang X., Peng Z., Li Y., Lin Y., Liang L., Wang G., Chen Y., Chen Q., Pan T., He X, & Zhang H. (2020). Nanoparticle Vaccines Based on the Receptor Binding Domain (RBD) and Heptad Repeat (HR) of SARS-CoV-2 Elicit Robust Protective Immune Responses. Immunity, 53(6), 1315-1330.e9.

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SARS-CoV-2 Variant RBD Binding Kinetics

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The measurements of BA.1_RBD‐N395‐403‐NP, BA.2_RBD‐N316‐333‐NP, BA.5_RBD‐N360‐370‐NP, BA.2.75_RBD‐S539‐546‐NP, Delta _RBD‐S1136‐1155NP, D614G_RBD‐S865‐873‐NP, and Mosaic_RBD‐NP binding to hACE2 were carried out with a BIAcoreT100 instrument (GE Healthcare). The BIAcore CM5 sensor chip and the amine‐coupling kit were purchased from GE Healthcare. hACE2 was attached to a CM5 sensor chip (carboxymethylated dextran covalently attached to a gold surface) with an amine coupling kit (GE Healthcare). BA.1_RBD‐N395‐403‐NP, BA.2_RBD‐N316‐333‐NP, BA.5_RBD‐N360‐370‐NP, BA.2.75_RBD‐S539‐546‐NP, Delta _RBD‐S1136‐1155‐NP, D614G_RBD‐S865‐873‐NP, and Mosaic_RBD‐NP were diluted to different concentrations before injection (30 µL min⁻¹). Each protein was monitored for about 120 s of hACE2‐binding protein. The running buffer was cycled with a dissociation time of 200 s. The Biacore T100 instrument was used to record the signal according to standard protocol.

Zhang X., Wu S., Liu J., Chen R., Zhang Y., Lin Y., Xi Z., Deng J., Pu Z., Liang C., Feng J., Li R., Lin K., Zhou M., Liu Y., Zhang X., Liu B., Zhang Y., He X, & Zhang H. (2023). A Mosaic Nanoparticle Vaccine Elicits Potent Mucosal Immune Response with Significant Cross‐Protection Activity against Multiple SARS‐CoV‐2 Sublineages. Advanced Science, 10(27), 2301034.

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Kinetic Analysis of RNase E Binding

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E. coli and M. tuberculosis RNase E were immobilized covalently using amine coupling to the surface of a Biacore CM5 sensor chip (GE Healthcare). Immobilization levels were approximately 1500 RUs. Assessment of compound binding were conducted by injecting varying compound concentrations in PBS containing 5% DMSO at flow rates of 30–90 μl/min for ~60 s over the reference and test flow cells. A Biacore T100 instrument was used and the data collected was reference and buffer subtracted prior to steady state analysis using data fitting functions provided in the Biacore T100 Evaluation Software.

Kime L., Vincent H.A., Gendoo D.M., Jourdan S.S., Fishwick C.W., Callaghan A.J, & McDowall K.J. (2015). The First Small-Molecule Inhibitors of Members of the Ribonuclease E Family. Scientific Reports, 5, 8028.

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Biacore Analysis of ADP-ribose Binding

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Recombinant RCD1-His and GST-RCD1ΔWWE proteins were coupled to a Biacore CM5 sensor chip (GE Healthcare) via amino-groups. PAR (625 nM) (Trevigen) was profiled at a flow rate of 30 mL/min for 300 s, followed by 600 s flow of wash buffer (10 mM HEPES, pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.05% Surfactant P20). Mono ADP-ribose (Sigma) and cyclic ADP-ribose (Sigma) were profiled at 1 mM concentration. After analysis in BiaEvalution (Biacore, GE Healthcare), the normalized resonance units were plotted over time with the assumption of one-to-one binding.

Vainonen J.P., Gossens R., Krasensky-Wrzaczek J., De Masi R., Danciu I., Puukko T., Battchikova N., Jonak C., Wirthmueller L., Wrzaczek M., Shapiguzov A, & Kangasjärvi J. (2023). Poly(ADP-ribose)-binding protein RCD1 is a plant PARylation reader regulated by Photoregulatory Protein Kinases. Communications Biology, 6, 429.

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Kinetic analysis of fibrillin-ADAMTSL2 interaction

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Purified fibrillin peptides and ADAMTSL2 were diluted in 10 mM MES buffer, pH 6.0 and immobilized on a BIAcore CM5 sensor chip (research grade, GE Healthcare, Piscataway, NJ) with the amine coupling kit according to the manufacturer's instructions. About 1500 resonance units were coupled to the chip for analysis in a BIAcore 3000 instrument (GE Healthcare, Piscataway, NJ). The kinetic analysis was performed at 25°C in 10 mM HEPES buffer, pH 7.4 including 150 mM NaCl, 2 mM CaCl₂ and 0.005% (v/v) surfactant P20 (running buffer) at a flow rate of 20 μl/min. Purified recombinant fibrillin-1 and -2 fragments, as described previously (Jensen et al., 2001 (link); Lin et al., 2002 (link)), were diluted in running buffer at different concentrations and injected through an uncoupled control flow cell and the flow cell coupled to ADAMTSL2 (Koo et al., 2007 (link)). Association was allowed for 3-6 min followed by a 10-min dissociation phase. 1 M NaCl with 2–10% (v/v) acetonitrile was used for regeneration after each injection at a flow rate of 50 μl/min for 30-60 s. The stabilization time following the regeneration was 2 min.

Hubmacher D., Wang L.W., Mecham R.P., Reinhardt D.P, & Apte S.S. (2015). Adamtsl2 deletion results in bronchial fibrillin microfibril accumulation and bronchial epithelial dysplasia – a novel mouse model providing insights into geleophysic dysplasia. Disease Models & Mechanisms, 8(5), 487-499.

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