T200 instrument

Manufactured by Cytiva

Sourced in United States, China

The T200 instrument is a piece of lab equipment designed for label-free, real-time molecular interaction analysis. It provides accurate and sensitive measurements of biomolecular interactions, such as protein-protein, protein-small molecule, and protein-nucleic acid interactions.

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50 protocols using t200 instrument

SPR Analysis of ACE2-Spike Interaction

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To confirm that the in vitro produced proteins (ACE2 and spike-f) interact with each other, an affinity measurement experiment was performed utilising SPR. Spike-f was immobilized via amine coupling on a CM5 chip (Cytiva) at a flow rate of 10 μl/min to 750 RU. A multicycle kinetic analysis was performed on a T200 instrument (Cytiva) using PBS supplemented with 0.05 % (v/v) Tween20 (PBS-T) as running buffer. ACE2 was applied in three different concentrations (250 nM, 125 nM and 62.5 nM) and the purified antibodies were analysed in three concentrations (4 μM, 2 μM and 1 μM). ACE2 and the antibodies were allowed to associate with the spike-based proteins for 400 s before dissociation was monitored over 600 s at a flow rate of 30 μl/min. The kinetic parameters were determined using the Biacore T200 Evaluation Software (Cytiva) and a 1:1 binding model. All runs were followed by regeneration using 10 mM HCl at 30 μl/min for 30 s.

Mravinacova S., Jönsson M., Christ W., Klingström J., Yousef J., Hellström C., Hedhammar M., Havervall S., Thålin C., Pin E., Tegel H., Nilsson P., Månberg A, & Hober S. (2021). A cell-free high throughput assay for assessment of SARS-CoV-2 neutralizing antibodies. New Biotechnology, 66, 46-52.

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SARS-CoV-2 Spike Protein Binding Kinetics

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SPR binding measurements were performed using a Biacore T200 instrument, running buffer was Cytiva HBS-EP+ (pH 7.4), and all measurements were performed at 25 °C. For Fab binding, anti-AviTag pAb (for capturing S proteins) or StrepTactin XT (for capturing RBDs) were covalently immobilized on CM5 chips. S2X259 Fab concentrations were 11, 33, 100, and 300 nM run as single-cycle kinetics. Double reference-subtracted data were fit to a 1:1 binding model using Biacore Evaluation software, which yields an “apparent KD” for the S-binding data because the kinetics also reflect S conformational dynamics. For SARS-CoV-2 S the dissociation rate was too slow to fit, so the K_D,app is reported as an upper limit. The K_D above 1 μM is approximate and was determined from a fit where Rmax was set to a constant based on theoretical Rmax.

Tortorici M.A., Czudnochowski N., Starr T.N., Marzi R., Walls A.C., Zatta F., Bowen J.E., Jaconi S., Di Iulio J., Wang Z., De Marco A., Zepeda S.K., Pinto D., Liu Z., Beltramello M., Bartha I., Housley M.P., Lempp F.A., Rosen L.E., Dellota E J.r., Kaiser H., Montiel-Ruiz M., Zhou J., Addetia A., Guarino B., Culap K., Sprugasci N., Saliba C., Vetti E., Giacchetto-Sasselli I., Fregni C.S., Abdelnabi R., Foo S.Y., Havenar-Daughton C., Schmid M.A., Benigni F., Cameroni E., Neyts J., Telenti A., Virgin H.W., Whelan S.P., Snell G., Bloom J.D., Corti D., Veesler D, & Pizzuto M.S. (2021). Broad sarbecovirus neutralization by a human monoclonal antibody. Nature, 597(7874), 103-108.

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Kinetic Analysis of LmFPPS Binding

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The binding affinities of LmFPPS for FPP and GPP were measured using surface plasmon resonance (SPR) with a Biacore T200 instrument (Cytiva) at 25°C. WT-LmFPPS was diluted to 10 μg/ml in a buffer consisting of 25 mM HEPES pH 7.5, 5 mM MgCl₂, 300 mM NaCl, 1 mM DTT and 0.05% Tween 20. The WT protein was then immobilized onto flow cell 2 (Fc2) of a Biacore CM5 (carboxymethyl-dextran) sensor chip via amine coupling to approximately 5000 resonance units (RU). Binding of analytes FPP and GPP were measured with multi-cycle kinetics. FPP was injected over immobilized LmFPPS at four increasing concentrations: 1.5, 6.2, 25 and 100 μM. Similarly, GPP was separately injected at varying concentrations ranging from 0.1 to 25 μM. The mutants were analyzed using the same protocol as the WT-LmFPPS. Reference subtracted and blank subtracted binding curves were fitted into a 1:1 interaction model and the steady-state affinity ‘K_D’ (equilibrium dissociation constant) was determined from steady-state affinity analysis using Biacore T200 Evaluation Software v3.2 (Cytiva).

Maheshwari S., Kim Y.S., Aripirala S., Murphy M., Amzel L.M, & Gabelli S.B. (2020). Identifying structural determinants of product specificity in Leishmania major farnesyl diphosphate synthase. Biochemistry, 59(29), 2751-2759.

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Kinetic Analysis of RBD-Fab Binding

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Measurements were performed using a Biacore T200 instrument. A CM5 chip with covalently immobilized StrepTactin XT was used for surface capture of Twin-Strep Tag containing RBDs. Running buffer was HBS-EP+ pH 7.4 (Cytiva) and measurements were performed at 25 LJC. Experiments were performed with a 3-fold dilution series of monomeric S2X324 Fab: 300, 100, 33, 11 nM and were run as single-cycle kinetics. Data were double reference-subtracted and fit to a binding model using Biacore Evaluation software. The 1:1 binding model was used to estimate the kinetics parameters. The experiment was performed twice with two biological replicates for each ligand (RBDs). KD values were reported as the average of two replicates with the corresponding standard deviation.

Park Y.J., Pinto D., Walls A.C., Liu Z., Marco A.D., Benigni F., Zatta F., Silacci-Fregni C., Bassi J., Sprouse K.R., Addetia A., Bowen J.E., Stewart C., Giurdanella M., Saliba C., Guarino B., Schmid M.A., Franko N., Logue J., Dang H.V., Hauser K., Iulio J.D., Rivera W., Schnell G., Rajesh A., Zhou J., Farhat N., Kaiser H., Montiel-Ruiz M., Noack J., Lempp F.A., Janer J., Abdelnabi R., Maes P., Ferrari P., Ceschi A., Giannini O., de Melo G.D., Kergoat L., Bourhy H., Neyts J., Soriaga L., Purcell L.A., Snell G., Whelan S.P., Lanzavecchia A., Virgin H.W., Piccoli L., Chu H., Pizzuto M.S., Corti D, & Veesler D. (2022). Imprinted antibody responses against SARS-CoV-2 Omicron sublineages. bioRxiv.

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Antibody Competition Reversal Assay

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For the reversal of growth inhibition experiments, we determined the concentrations of E3 and gAMA1 that completely blocked the E3-specifc and gAMA1-specifc antibodies. The antigens were buffer exchanged against RPMI 1640 (PAA, E15-041) with HEPES without L-glutamine by repeated rounds of dilution and concentration using a VivaSpin 15R column with a MWCO of 10 kDa. Total IgG preparations from the third bleed (day 91) were diluted to a final concentration in the competition mix resulting in 40–70% inhibition in a standard growth inhibition assay, and incubated overnight at 4°C with either gAMA1 (final concentrations 120, 12 and 1.2 μg/ml) or E3 (final concentrations 1230, 123 and 12.3 μg/ml). The competition mix was analyzed against the corresponding antigen (E3 or gAMA1) immobilized on the CM5-S-Series sensor chips using a Biacore T200 instrument. Full competition was defined as a RU signal less than 5% of the sample containing no competitor.

Boes A., Spiegel H., Voepel N., Edgue G., Beiss V., Kapelski S., Fendel R., Scheuermayer M., Pradel G., Bolscher J.M., Behet M.C., Dechering K.J., Hermsen C.C., Sauerwein R.W., Schillberg S., Reimann A, & Fischer R. (2015). Analysis of a Multi-component Multi-stage Malaria Vaccine Candidate—Tackling the Cocktail Challenge. PLoS ONE, 10(7), e0131456.

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Surface Plasmon Resonance Binding Assays

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Surface plasmon resonance experiments were performed with a Biacore T200 instrument using Series S CM5 sensor chips as previously described^{14 (link)}. For S3–4 and DMF5 binding experiments, S3–4 or DMF5 was immobilized on the sensor chip at 1000–3000 response units via standard amine coupling and peptide/MHC injected as analyte. Steady-state experiments were performed at 25 °C at a flowrate of 10 μL/min, with responses subtracted from an activated then deactivated blank flow cell. To test if S3–4 binds peptide/MHC in a peptide independent manner, A6 c134 was immobilized at a chip density of ~3000 RU. Every sample injection cycle consisted of 20 μM Tax/HLA-A2 to maintain the TCR-peptide/MHC complex on the surface, followed by 500 seconds of buffer, then varying concentrations of S3–4, with responses again corrected for responses from a blank flow cell. SPR data was processed in BiaEvaluation 4.1 and analyzed in OriginPro 9.0 using a 1:1 binding model and global fitting^{15 (link)}. The kinetic titration of S3–4_MQ binding to Tax/HLA-A2 was performed as previously described¹⁶. Briefly, the TCR was coupled to a sensor surface at approximately 500 response units. A series of five injections of increasing concentration of Tax/HLA-A2 were used at a flow rate of 30 μL/min at 25°C. Data were fit with a 1:1 kinetic titration model with drift using BIAevaluation 4.1.

Singh N.K., Alonso J.A., Harris D.T., Anderson S.D., Ma J., Hellman L.M., Rosenberg A.M., Kolawole E.M., Evavold B.D., Kranz D.M, & Baker B.M. (2020). An engineered T cell receptor variant realizes the limits of functional binding modes. Biochemistry, 59(43), 4163-4175.

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Measuring HAdV Fiber Knob-CAR Interactions

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All measurements of HAdV long fiber knobs:CAR interactions were performed at 25°C using a Biacore T-200 instrument. CAR (CXADR Fc chimera full-length extracellular D1D2 domain; R&D Systems, Minneapolis, MN, USA) was immobilized to a CM5 chip, using the Amine Coupling Kit (GE Healthcare), to a concentration of 1.2 to 1.5 ng·mm² (∼1,500 response units). All binding assays were performed at 25°C using running buffer [10 mM phosphate buffer (pH 7.4), 140 mM NaCl, 0.27 mM KCl, and 0.05% Tween20]. The analytes were diluted in running buffer and serially diluted in running buffer in a twofold concentration dilution (2000 to 7.81125 nM) and then injected in series over the reference and experimental biosensor surfaces for 120 s at a flow rate of 30 µL/min. Blank samples containing only running buffer were also injected under the same conditions to allow for double referencing. After each cycle, the biosensor surface was regenerated with a 60-s pulse of 10 mM glycine (pH 1.5) at a flow rate of 30 µL/min.

Becker M., Conca D.V., Dorma N., Mistry N., Hahlin E., Frängsmyr L., Bally M., Arnberg N, & Gerold G. (2023). Efficient clathrin-mediated entry of enteric adenoviruses in human duodenal cells. Journal of Virology, 97(10), e00770-23.

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Biacore Analysis of RBD-mFc Binding

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Compound solutions with a series of increasing concentrations were applied to all four channels at a 30 μL/min flow rate. RBD-mFc was immobilized on a CM5 sensor chip using standard amine-coupling with running buffer HBS-EP +using a Biacore T200 instrument. The RBD-mFc was immobilized to flow channels 2, and immobilization levelswas ∼3800 RU. The resulting data were fit to a 1:1 binding model using Biacore T200 evaluation software 2.0.

Ma L., Li Y., Shi T., Zhu Z., zhao J., Xie Y., Wen J., Guo S., Wang J., Ding J., Liang C., Shan G., Li Q., Ge M, & Cen S. (2023). Teicoplanin derivatives block spike protein mediated viral entry as pan-SARS-CoV-2 inhibitors. Biomedicine & Pharmacotherapy, 158, 114213.

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SPR Analysis of eEF2 Binding

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SPR was performed using the Biacore T200 instrument as previously described [22 (link)]. eEF2 protein was diluted in 10 mM sodium acetate (pH 4.5) at a concentration of 15 μg/mL for immobilization. After immobilizing about 4 µg eEF2 protein on a CM5 chip (Cat#BR-1005–30, GE Healthcare), TSN or DDD107498 (dissolved in PBS with 0.1% DMSO) were perfused through the ligand channels at different concentrations ranging from 10 nM to 1 µM. The T200 evaluation software was used to analyze the binding affinity between eEF2 with TSN or DDD107498.

Jia X., Wang P., Huang C., Zhao D., wu Q., Lu B., Nie W., Huang L., Tian X., li P., Laster K.V., Jiang Y., Li X., Li H., Dong Z, & Liu K. (2023). Toosendanin targeting eEF2 impedes Topoisomerase I & II protein translation to suppress esophageal squamous cell carcinoma growth. Journal of Experimental & Clinical Cancer Research : CR, 42, 97.

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Membrane Binding Kinetics of Cyclotides

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Synthetic lipids POPC and POPE were purchased from Avanti Polar Lipids. Two model membranes were prepared: POPC and POPC/POPE (80:20, mol/mol) using the method described previously (61 (link)). Briefly, small unilamellar vesicles resuspended in Hepes buffer (10 mM Hepes, 150 mM NaCl, pH 7.4, filtered) were obtained using a mini extruder (Avanti) with a 50 nm polycarbonate membrane. Membrane binding of acyclotides and cyclotides toward POPC or POPC/POPE was evaluated using an L1 sensor chip on a Biacore T200 instrument. A suspension of small unilamellar vesicles (0.5 mM) was deposited onto an L1 chip surface at 2 μl/min for 40 min, followed by a short pulse of 10 mM NaOH. The cyclotide solutions were injected over the lipid surface at 5 μl/min for 180 s, and the dissociation process was recorded for 10 min. Dose–response curves were established through the calculated peptide-to-lipid molar ratio to evaluate the binding ability of the tested peptides. K_D was calculated by fitting in specific binding with Hill slope using GraphPad Prism software (GraphPad Software Inc) (61 (link)).

Dang T.T., Huang Y.H., Ott S., Harvey P.J., Gilding E.K., Tombling B.J., Chan L.Y., Kaas Q., Claridge-Chang A, & Craik D.J. (2022). The acyclotide ribe 31 from Rinorea bengalensis has selective cytotoxicity and potent insecticidal properties in Drosophila. The Journal of Biological Chemistry, 298(10), 102413.

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