Biacore s200

Manufactured by GE Healthcare

Sourced in United States, Sweden, United Kingdom, China

The Biacore S200 is a label-free interaction analysis system designed for biomolecular interaction studies. It offers real-time monitoring of molecular interactions, enabling the measurement of affinity, kinetics, and concentration of biomolecular complexes.

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Biacore S200 instrument (10 citations) Biacore S200 system (9 citations) Biacore S200 evaluation software (7 citations) Biacore S200 machine (3 citations) BIAcore S200 SPR instrument (3 citations) Biacore S200 Evaluation Software Version 1.1 (2 citations) Biacore S200 Evaluation Software 1.0 (2 citations) Biacore S200 analysis software (2 citations)

68 protocols using biacore s200

Quantifying CDK2-cyclin A Interactions

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SPR analysis was carried out using a Biacore S200 (GE Healthcare). The sensor chip CM5 surface was functionalized using an anti GST tag antibody by amine-coupling. Routinely, ligand capture on flow cell number 2 was carried out for 60 s followed by multiple buffer injection to remove the excess of inactive protein, and the intensity was normalized against flow cell number 1 (GST control as ligand to correct for non-specific interactions and bulk effects). Experiments were performed in HEPES 20 mM pH7.4, NaCl 150 mM Tween20 0.01% at 20 °C. GSTp27M was captured on the functionalized surface of flow cell 2 for 60 s at a flowrate of 5 µl min⁻¹. CDK2-cyclin A wild-type and mutants were then titrated as analytes using a 4-fold dilution series and then the chip was regenerated using glycine pH 2.1 for 60 s. The sample contact time (60 s) was measured using high performance settings followed by 240 s of dissociation time at 30 µl min⁻¹. Two independent (biological replicate) experiments were performed. Sensorgrams were analyzed using Biacore S200 Evaluation Software 1.0 (GE Healthcare) and the curves were fitted using 1:1 binding kinetics.

Salamina M., Montefiore B.C., Liu M., Wood D.J., Heath R., Ault J.R., Wang L.Z., Korolchuk S., Baslé A., Pastok M.W., Reeks J., Tatum N.J., Sobott F., Arold S.T., Pagano M., Noble M.E, & Endicott J.A. (2021). Discriminative SKP2 Interactions with CDK-Cyclin Complexes Support a Cyclin A-Specific Role in p27KIP1 Degradation. Journal of Molecular Biology, 433(5), 166795.

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SPR Binding Kinetics of CAIX Inhibitor

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Surface
plasmon resonance (SPR) experiments were performed at room temperature
using a Biacore S200 instrument (GE Healthcare). CM5 chips (Series
S) and filtered phosphate-buffered saline (PBS) pH 7.4 with dimethyl
sulfoxide (DMSO) (5% v/v) as a flow buffer were used for all experiments.
Human CAIX was immobilized on the chip to 500 response units (R.U.)
using EDC·HCl and NHS according to the manufacturer’s
instructions. Serial dilutions of compound 7 (AAZ⁺-ValCit-Cry55gly) in a running buffer at a flow rate of 20
μL/min were used as analytes. The chip surface was regenerated
after each cycle by a short treatment with DMSO (50% v/v) in PBS.
Sensorgrams were solvent-corrected and the binding kinetics was analyzed
with the Biacore S200 evaluation software using the 1:1 Langmuir binding
model.

Cazzamalli S., Figueras E., Pethő L., Borbély A., Steinkühler C., Neri D, & Sewald N. (2018). In Vivo Antitumor Activity of a Novel Acetazolamide–Cryptophycin Conjugate for the Treatment of Renal Cell Carcinomas. ACS Omega, 3(11), 14726-14731.

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Quantifying MERS-CoV S1 Binding Kinetics

Cited 1 time

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The binding between Nbs and MERS-CoV S1 or RBD protein was detected using a BiacoreS200 instrument (GE Healthcare) as previously described (29 (link)). Briefly, recombinant Fc-fused MERS-CoV RBD-Fc protein or NbMS10-Fc Nb (5 μg/ml) was captured using a Sensor Chip protein A (GE Healthcare), and recombinant His₆-tagged MERS-CoV S1-His protein or NbMS10 Nb at various concentrations was flown over the chip surface in a running buffer containing 10 mM HEPES (pH 7.4), 150 mM NaCl, 3 mM EDTA, and 0.05% surfactant P20. The sensorgram was analyzed using Biacore S200 software, and the data were fitted to a 1:1 binding model.

Zhao G., He L., Sun S., Qiu H., Tai W., Chen J., Li J., Chen Y., Guo Y., Wang Y., Shang J., Ji K., Fan R., Du E., Jiang S., Li F., Du L, & Zhou Y. (2018). A Novel Nanobody Targeting Middle East Respiratory Syndrome Coronavirus (MERS-CoV) Receptor-Binding Domain Has Potent Cross-Neutralizing Activity and Protective Efficacy against MERS-CoV. Journal of Virology, 92(18), e00837-18.

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Mouse SAA1 Binding Affinity to MD2, TLR4

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The binding affinity of mouse SAA1 to MD2 or TLR4 was determined by surface plasmon resonance measurements using a BIAcore S200 instrument (GE healthcare). Mouse SAA1 were prepared as mentioned above. Mouse TLR4 was purchased from R&D system, and mouse MD2 was purchased from Beijing ABIOCENTER Co., Ltd. For the binding affinity of mouse SAA1 to MD2, SAA1 was immobilized on the sensor surface. Different concentrations of MD2 were allowed to run sequentially over the immobilized SAA1 to monitor the functionality of the protein surface. For the binding affinity of mSAA1 to mTLR4, TLR4 was immobilized on the sensor surface. Different concentrations of SAA1 were injected over the immobilized TLR4 and reference surface with at least 30 s association and dissociation times. Surface regeneration was achieved using dissociation for a time period allowing the response to return to baseline. SPR equilibrium binding data, consisting of Req values from 6-point concentration series, were analyzed by fitting a steady model to yield Rmax and Kd values using BIAcore S200 Evaluation Software.

Jiang B., Wang D., Hu Y., Li W., Liu F., Zhu X., Li X., Zhang H., Bai H., Yang Q., Yang X., Ben J, & Chen Q. (2022). Serum amyloid A1 exacerbates hepatic steatosis via TLR4-mediated NF-κB signaling pathway. Molecular Metabolism, 59, 101462.

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SPR Analysis of Kelch Domain Binding

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Assays were performed at 25°C using a BIACORE S200 (GE Healthcare) surface plasmon resonance (SPR) instrument. The Kelch domains of KLHL20 and KLHL3 were immobilized on sensor chip CM5 (GE Healthcare) using amine coupling. Reference flow cells had no immobilized protein. Binding was monitored using a flow rate of 30 μL/min. The peptide analytes were prepared in HBS-P buffer (GE Healthcare). Data reported were after reference flow cell signal subtraction. Data were analyzed by one-site steady-state affinity analysis using the BIACORE S200 Evaluation software and the fitting equation

R e q = \frac{C R m a x}{K D + C} + R I

. (RI, bulk refractive index contribution; R_max, maximum response; K_D, dissociation constant; C, analyte concentration). Peptides were obtained from Severn Biotech Ltd.

Chen Z., Picaud S., Filippakopoulos P., D'Angiolella V, & Bullock A.N. (2019). Structural Basis for Recruitment of DAPK1 to the KLHL20 E3 Ligase. Structure(London, England:1993), 27(9), 1395-1404.e4.

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Biacore-Based Protein-Peptide Binding Assay

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Assays were conducted using a Biacore S200 (GE Healthcare Bio-Sciences AB, SE-751 84, Uppsala, Sweden). All reagent coupling kits and sensors were from GE Healthcare. All reagents were from Sigma-Aldrich (3300 S. Second St., St. Louis, MO 63118, USA) unless otherwise stated. The MBB-tagged target were expressed recombinantly and purified in-house using standard protocols. All experiments were performed using a buffer containing 50 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, (HEPES), pH 7.5, containing 0.15 M sodium chloride and 0.2 mM tris(2-carboxyethyl)phosphine (TCEP). The 3,879 Da peptide was synthesized by GenScript (860 Centennial Ave, Piscataway, NJ 08854, USA) and purity was >95% by HPLC.

Quinn J.G., Steffek M., Bruning J.M., Frommlet A, & Mulvihill M.M. (2019). Unlocking latent kinetic information from label-free binding. Scientific Reports, 9, 18389.

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Characterization and Bioactivity of F8-IL2 Fusion

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F8-IL2 was analyzed using SDS-PAGE in non-reducing and reducing conditions, size exclusion chromatography (Superdex 200 Increase, 10/300 GL, GE Healthcare), and surface plasmon resonance analysis (BIAcore S200, GE Healthcare) on an EDA antigen-coated CM5 BIAcore sensor chip (GE Healthcare). The biological activity of F8-IL2 and IL2 (Proleukin, Roche) was determined as described before (9 (link)). Briefly, 10x10⁶cultured CTLL-2 cells were starved in CTLL2 culture medium (RPMI 1640 (Gibco) supplemented with 10% FBS, 1 % Ultraglutamine, 25 mM Hepes (Gibco), 0.05 mM β-mercaptoethanol (Sigma Aldrich)) without IL2 for 24 hours. Starved CTLL-2 cells (2x 10⁴/well) were seeded in 96-well plates in CTLL2 culture medium containing varying concentrations (5x10^-12 – 10^-9 M IL2 equivalents) of IL2 equivalents (F8-IL2 or as positive control purchased human IL2 (Roche Diagnostics) in triplicates. After 48 hours at 37°C, cell proliferation was determined with the Cell Titer 96^® Aqueous One Solution (Promega) according to the manufacturer’s instructions by measuring the OD at 490 nm and 620 nm. Percent proliferation was calculated as follows: % proliferation = (OD_490-62^treated-OD_490-620^medium)/(OD_490-62^untreated-OD_490-620^medium) x 100 %.

Hutmacher C., Núñez N., Liuzzi A.R., Becher B, & Neri D. (2019). Targeted delivery of IL2 to the tumor stroma potentiates the action of immune checkpoint inhibitors by preferential activation of NK and CD8+ T cells. Cancer immunology research, 7(4), 572-583.

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SPR-based Ligand Binding Assay for BRD4

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SPR-based ligand binding
assays were performed using a BIAcore S200 (GE Healthcare) at 25 °C
using single cycle affinity. Immobilization of BRD4 was achieved using
standard amine coupling on a CM5 chip surface. The surface was prepared
through activation with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide/N-hydroxy succinimide (EDC/NHS), followed by injection of
10 μg mL^–1 BRD4 until a target level of 8000
RU was reached. The surface was then quenched using 1 M ethanolamine
and washed with running buffer (10 mM HEPES, 150 mM NaCl, 0.01% (v/v)
Tween-20, 0.5 mM TCEP, and 1% (v/v) DMSO) at a flow rate of 30 μL
min^–1. XMD8-92 and 46 were injected in a dose–response
manner (nine points ranging from 0 to 20 μM) with a contact
time of 30 s and a dissociation time of 160 s in series across the
reference and BRD4-immobilized flow cells using solvent correction
to account for bulk refractive index changes. The reference channel
response was subtracted from the BRD4-immobilized channel response,
and dose–response data were fitted using an affinity steady-state
1:1 binding model to determine the K_d.

Miller D.C., Reuillon T., Molyneux L., Blackburn T., Cook S.J., Edwards N., Endicott J.A., Golding B.T., Griffin R.J., Hardcastle I., Harnor S.J., Heptinstall A., Lochhead P., Martin M.P., Martin N.C., Myers S., Newell D.R., Noble R.A., Phillips N., Rigoreau L., Thomas H., Tucker J.A., Wang L.Z., Waring M.J., Wong A.C., Wedge S.R., Noble M.E, & Cano C. (2022). Parallel Optimization of Potency and Pharmacokinetics Leading to the Discovery of a Pyrrole Carboxamide ERK5 Kinase Domain Inhibitor. Journal of Medicinal Chemistry, 65(9), 6513-6540.

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Transient Expression and Purification of Fusion Proteins in CHO-S Cells

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All fusion proteins were expressed using polyethyleneimine (PEI)-mediated transient gene expression in CHO-S cells.(Hacker et al., 2013 (link)) For 1 mL of production 10⁶ CHO-S cells in suspension were centrifuged and resuspended in 0.5 mL ProCHO4. 0.9 μg of desired plasmid DNA followed by 2.5 μg of PEI (Polysciences) was added to the cells and the suspension gently mixed. The cells were incubated for 6 days in a shaking incubator at 31°C. The procedure was scaled up in order to obtain the desired production volume.
The fusion proteins were purified from the culture medium via protein A affinity chromatography and analyzed using SDS-PAGE in non-reducing and reducing conditions, Size exclusion chromatography (Superdex 200 increase, 10/300 GL) (GE Healthcare, UK) and surface plasmon resonance analysis (BIAcore S200) (GE Healthcare, UK) on an EDA antigen-coated CM5 Biacore sensor chip.

Schmid A.S., Tintor D, & Neri D. (2018). Novel antibody-cytokine fusion proteins featuring granulocyte-colony stimulating factor, interleukin-3 and interleukin-4 as payloads. Journal of biotechnology, 271, 29-36.

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Kinetic Analysis of ATG12-Compound Binding

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For the surface plasmon resonance experiments, Biacore S200 (GE Healthcare, Sweden) was used. For the preparation of the ATG12-Flag immobilized sensor chip, purified ATG12-Flag in HEPES buffer (50 mM HEPES [Bio-Lab Ltd, 773,233,100], 50 mM NaCl, and 3 mM β-mercaptoethanol) was injected upon a Series S Sensor Chip CM5 (Cytiva, GE Healthcare, 14100530S). The running buffer contained 50 mM HEPES, 50 mM NaCl, 3 mM β-mercaptoethanol, and 5% DMSO (Sigma-Aldrich, D2650). Compound 189 was diluted in running buffer as indicated and injected over the immobilized ATG12-Flag sensor chip at 50 uL/min rate. Association was carried out for 1 min, and dissociation was measured at a flow rate of 50 μl/min at 25°C. No regeneration was required as the sensogram reached baseline after 1 min. The binding and dissociation of compound 189 to ATG12 was too fast to determine Kd. As 5% DMSO was included in the samples as well as the running buffer, solvent correction was used in order to eliminate bulk responses from solvent effects.

Nuta G.C., Gilad Y., Goldberg N., Meril S., Bahlsen M., Carvalho S., Kozer N., Barr H., Fridmann Sirkis Y., Hercík K., Břehová P., Nencka R., Bialik S., Eisenstein M, & Kimchi A. (2023). Identifying a selective inhibitor of autophagy that targets ATG12-ATG3 protein-protein interaction. Autophagy, 19(8), 2372-2385.

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