Octet red96e system

Manufactured by Sartorius

Sourced in Germany

The Octet RED96e system is a label-free, real-time biomolecular interaction analysis instrument. It is designed for the quantitative analysis of biomolecular interactions, including protein-protein, protein-small molecule, and protein-nucleic acid interactions. The Octet RED96e system uses bio-layer interferometry technology to measure the association and dissociation kinetics and affinities of these interactions.

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Lab products found in correlation

Sulfo nhs lc lc biotin, by Thermo Fisher Scientific (2 mentions) Zeba spin desalting columns 7k mwco 0.5 ml, by Thermo Fisher Scientific (2 mentions) Ni nta fast start kit, by Qiagen (1 mentions) Amicon ultra 15 centrifugal filter unit, by Merck Group (1 mentions) Ds 11 fx, by DeNovix (1 mentions) Tween 20, by Bio-Rad (1 mentions) Streptavidin coated biosensors, by Sartorius (1 mentions) Fentanyl citrate, by Merck Group (1 mentions) Peaq itc, by Malvern Panalytical (1 mentions) Octet data analysis ht 12, by Sartorius (1 mentions) Hbs ep buffer, by Cytiva (1 mentions) Human igg1 fc, by R&D Systems (1 mentions)

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Octet RED96e (21 citations) Octet system (4 citations)

12 protocols using octet red96e system

Kinetic Analysis of RBD-Specific Antibodies

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Analysis of binding kinetics of RBD-specific antibodies in serum samples were performed using the Octet RED96e system (Sartorius) as per the manufacturer’s recommendations. Purified RBDs of WT, Delta, and Omicron were biotinylated with Sulfo-NHS-LC-LC-Biotin (Thermo Fisher Scientific) in 5 molar excess at ambient temperature for 30 minutes. Excess of biotin was removed by size exclusion chromatography using Zeba Spin Desalting Columns 7K MWCO 0.5 mL (Thermo Fisher Scientific) according to the manufacturer’s protocol. Data were analyzed using the Octet Data Analysis HT 12.0 software applying the 1:1 fitting model for the dissociation step. The binding profile response of each sample is illustrated as the mean wavelength shift in nm. For affinity determination, the 1:1 global fit of the Data Analysis HT 12.0 software was used. Please consult the Supplementary Methods for full details.

Junker D., Becker M., Wagner T.R., Kaiser P.D., Maier S., Grimm T.M., Griesbaum J., Marsall P., Gruber J., Traenkle B., Heinzel C., Pinilla Y.T., Held J., Fendel R., Kreidenweiss A., Nelde A., Maringer Y., Schroeder S., Walz J.S., Althaus K., Uzun G., Mikus M., Bakchoul T., Schenke-Layland K., Bunk S., Haeberle H., Göpel S., Bitzer M., Renk H., Remppis J., Engel C., Franz A.R., Harries M., Kessel B., Lange B., Strengert M., Krause G., Zeck A., Rothbauer U., Dulovic A, & Schneiderhan-Marra N. (2022). Antibody Binding and Angiotensin-Converting Enzyme 2 Binding Inhibition Is Significantly Reduced for Both the BA.1 and BA.2 Omicron Variants. Clinical Infectious Diseases: An Official Publication of the Infectious Diseases Society of America, 76(3), e240-e249.

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Kinetic Analysis of Miro1-Specific Nanobodies

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Analysis of binding kinetics of Miro1-specific Nbs was performed using the Octet RED96e system (Sartorius) according to the manufacturer’s recommendations. In brief, 2–10 μg/ml solution of biotinylated Miro1-Nbs diluted in Octet buffer (HEPES, 0.1% BSA) was used for 40 s to immobilize the Nb on streptavidin coated biosensor tips (SA, Sartorius). In the association step, a dilution series of Miro1 ranging from 3.9 nM to 1 µM were reacted for 300 s followed by dissociation in Octet buffer for 720 s. Every run was normalized to a reference run using Octet buffer for association. Data were analyzed using the Octet Data Analysis HT 12.0 software applying the 1:1 ligand-binding model and global fitting.

Fagbadebo F.O., Kaiser P.D., Zittlau K., Bartlick N., Wagner T.R., Froehlich T., Jarjour G., Nueske S., Scholz A., Traenkle B., Macek B, & Rothbauer U. (2022). A Nanobody-Based Toolset to Monitor and Modify the Mitochondrial GTPase Miro1. Frontiers in Molecular Biosciences, 9, 835302.

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Quantification of rRBD Protein Using Octet RED96e

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The Octet RED96e system (Sartorius) was used for rRBD protein quantification in all supernatant samples using Anti-Penta-HIS (HIS1K) biosensors (Sartorius #18-5120). Supernatant samples were diluted 2- or 5-fold in phosphate buffered saline (PBS; Fisher Scientific) for analysis. HIS1K biosensors were hydrated for ≥ 10 min in a blend of production medium (Expi293 or BalanCD HEK293) and PBS to match the supernatant:PBS ratio in the sample matrix. During analysis, the sample plate was maintained at 30 °C and 1000 rpm. Sample concentrations were interpolated from an unweighted 4-parameter logistic (4PL) regression model for serial dilutions of purified rRBD internal standard material.
The internal standard was produced by transient transfection of the CAG-rRBD-wtEBNA1 plasmid into Expi293F cells as described above, purified using the Ni-NTA Fast Start Kit (Qiagen), and then buffer exchanged into PBS using Amicon Ultra-15 Centrifugal Filter Units (MilliporeSigma) with a 10 kilodalton (kDa) cutoff. Standard concentration was measured by A280 absorbance on a DS-11 FX+ (DeNovix) using a 32.6 Da molecular weight (un-glycosylated sequence estimate) and 33,350 M^− 1 cm^− 1 extinction coefficient.

Green E.A., Hamaker N.K, & Lee K.H. (2023). Comparison of vector elements and process conditions in transient and stable suspension HEK293 platforms using SARS-CoV-2 receptor binding domain as a model protein. BMC Biotechnology, 23, 7.

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Competitive Binding of Angiopoietins and 4E2

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An Octet Red 96e system (Sartorius, Goettingen) equipped with Ni-NTA sensors was used to measure the competitive binding of 4E2 and angiopoietins. The human anti-Tie2 antibody (50 nM)-immobilized biosensor was immersed in a well containing 100 nM ANGPT1 or ANGPT2 in phosphate-buffered saline (PBS) by centrifugation at 1,000 rpm for the first binding step. After the sensorgram plateaued (up to 2200 sec), the ANGPT1-bound sensor was moved to a well containing PBS buffer without a reagent, 100 nM ANGPT1 (or ANGPT2) or 27.2 nM 4E2 (the second binding step). To evaluate the competition between ANGPTs and 4E2, PBS without ANGPTs was also tested in the first binding step. Additionally, the first and second binding steps were reversed to evaluate the binding characteristics of 4E2.

Lee E., Lee E.A., Kong E., Chon H., Llaiqui-Condori M., Park C.H., Park B.Y., Kang N.R., Yoo J.S., Lee H.S., Kim H.S., Park S.H., Choi S.W., Vestweber D., Lee J.H., Kim P., Lee W.S, & Kim I. (2023). An agonistic anti-Tie2 antibody suppresses the normal-to-tumor vascular transition in the glioblastoma invasion zone. Experimental & Molecular Medicine, 55(2), 470-484.

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Quantification of PF4-Specific Antibody Binding

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Purified PF4 (ChromaTec, Greifswald, Germany) was biotinylated with Sulfo-NHS-LC-LC-Biotin (Thermo Fisher Scientific, Waltham, MA) in 5 molar excess at ambient temperature for 30 minutes. Excess biotin was removed by size exclusion chromatography using Zeba Spin Desalting Columns 7K MWCO 0.5 mL (Thermo Fisher Scientific) according to the manufacturer’s protocol. Analysis of binding kinetics of PF4-specific Abs in heat-inactivated serum samples or purified IgG fractions was performed using the Octet RED96e system (Sartorius, Goettingen, Germany) as per the manufacturer’s recommendations. Data were analyzed using the Octet Data Analysis HT 12.0 software applying the 2:1 heterogeneous ligand-binding model. For quantification, the averages of binding between 475 s and 480 s of the association step and 460 s and 462 s of the dissociation step was used to calculate the percent Residual Binding T_560s. The binding profile response of each sample is illustrated as the mean wavelength shift in nm. For details, see supplemental Material.

Singh A., Toma F., Uzun G., Wagner T.R., Pelzl L., Zlamal J., Freytag V., Weich K., Nowak-Harnau S., Rothbauer U., Althaus K, & Bakchoul T. (2022). The interaction between anti-PF4 antibodies and anticoagulants in vaccine-induced thrombotic thrombocytopenia. Blood, 139(23), 3430-3438.

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Binding Kinetics of rHA-ACE2 to SARS-CoV-2 S1

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Binding kinetics of rHA-ACE2 to biotinylated SARS-CoV-2 S1 protein (Acro Biosystems, #S1N-C82E8) was measured by Bio-Layer Interferometry (BLI) using an Octet Red 96e system (Sartorius). Soluble ACE2 (Sigma Aldrich, #SAE0064) was used as a control. The biotinylated SARS-CoV-2 S1 protein was immobilised on Streptavidin Dip and Read^TM sensor tips at a concentration of 8 nM in kinetics buffer (PBS supplemented with 0.02% Tween-20 and 0.1% bovine serum albumin (BSA)). For binding kinetics measurements rHA-ACE2 and soluble ACE2 samples were prepared in a 7-step two-fold dilution series starting at 3 µM and 125 nM, respectively, in kinetics buffer. Measurements were performed at 30 °C and 1000 rpm shaking with a 200 s association and 600 s dissociation step. Tips were regenerated between samples by alternating between 10 s incubation in 10 mM Glycine at pH 3 and kinetics buffer 6 times. SARS-CoV-2 S1 protein coated streptavidin sensors in kinetics buffer was used for baseline subtraction. Data analysis was performed using the Octet data analysis software (version 10.0.1.6) using a 1:1 interaction model curve fitting.

Fuchs E., Rudnik-Jansen I., Dinesen A., Selnihhin D., Mandrup O.A., Thiam K., Kjems J., Pedersen F.S, & Howard K.A. (2022). An albumin-angiotensin converting enzyme 2-based SARS-CoV-2 decoy with FcRn-driven half-life extension. Acta Biomaterialia, 153, 411-418.

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Kinetic Analysis of hSIRPα-Nbs Binding

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Analysis of binding kinetics of hSIRPα-specific Nbs was performed using the Octet RED96e system (Sartorius) as per the manufacturer’s recommendations. In brief, biotinylated hSIRPα (5 µg/mL) diluted in Octet buffer (PBS, 0.1% BSA, and 0.02% Tween-20) was immobilized on streptavidin coated biosensor tips (SA, Sartorius) for 40 s. In the association step, a dilution series of Nbs ranging from 0.625 nM to 320 nM were reacted for 240 s followed by dissociation in Octet buffer for 720 s. Every run was normalized to a reference run applying Octet buffer for association. Data were analyzed using the Octet Data Analysis HT 12.0 software applying the 1:1 ligand-binding model and global fitting. For epitope binning, two consecutive association steps with different Nbs were performed. By analyzing the binding behavior of the second Nb, conclusions about shared epitopes were drawn. For the hCD47 competition assay, hCD47 was biotinylated and immobilized on SA biosensors followed by the application of pre-mixed solutions containing hSIRPα (20 nM) and Nb (250 nM). hCD47-competing Ab KWAR23 (5 nM) was used as control.

Wagner T.R., Blaess S., Leske I.B., Frecot D.I., Gramlich M., Traenkle B., Kaiser P.D., Seyfried D., Maier S., Rezza A., Sônego F., Thiam K., Pezzana S., Zeck A., Gouttefangeas C., Scholz A.M., Nueske S., Maurer A., Kneilling M., Pichler B.J., Sonanini D, & Rothbauer U. (2023). Two birds with one stone: human SIRPα nanobodies for functional modulation and in vivo imaging of myeloid cells. Frontiers in Immunology, 14, 1264179.

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FcRn Binding Kinetics via Bio-Layer Interferometry

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An Octet Red96e system (Sartorius) was used for Bio-Layer Interferometry (BLI) to investigate FcRn binding kinetics. Streptavidin coated biosensors (Sartorius) were hydrated in PBS with 0.01% (v/v) Tween 20 (Bio-Rad) at pH 7.4 followed by immobilization of 8.75 nM biotinylated human FcRn (Immunitrack) in PBS with 0.01% (v/v) Tween 20 and then washed in fresh PBS with 0.01% (v/v) Tween 20. For all analytes [FcRn high-binding albumin (Albumin^HB) (Bern et al., 2020 (link)), LiTCo-Albu and LiTCo] a 2-fold dilution series starting from 200 nM was prepared in kinetics buffer (PBS with 25 mM CH₃COONa, 25 mM NaH₂PO₄, 150 mM NaCl, and 0.01% (v/v) Tween 20 at pH 5.5). For each analyte a control with no analyte but FcRn-coated biosensors and a control with 200 nM analyte but uncoated biosensors were also included. Binding kinetics were analyzed at 30°C. After a 180 s baseline in kinetics buffer, analyte association was performed in sample wells for 300 s before dissociation for 600 s in kinetics buffer, followed by biosensor regeneration in PBS with 0.01% (v/v) Tween 20 for 240 s. The control well without analyte was used for baseline subtraction. In the Octet System Data Analysis software (version 10.0.1.6) a 1:1 binding model was used to determine kinetic parameters.

Hangiu O., Compte M., Dinesen A., Navarro R., Tapia-Galisteo A., Mandrup O.A., Erce-Llamazares A., Lázaro-Gorines R., Nehme-Álvarez D., Domínguez-Alonso C., Harwood S.L., Alfonso C., Blanco B., Rubio-Pérez L., Jiménez-Reinoso A., Díez-Alonso L., Blanco F.J., Sanz L., Howard K.A, & Álvarez-Vallina L. (2022). Tumor targeted 4-1BB agonist antibody-albumin fusions with high affinity to FcRn induce anti-tumor immunity without toxicity. iScience, 25(9), 104958.

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Fentanyl Binding Kinetics Characterization

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Biolayer interferometry (BLI) was performed on an Octet Red 96e system (Sartorius). Streptavidin-coated biosensors were pre-hydrated with PBS-T (phosphate buffered saline pH 7.5, 0.05% Tween 20), and baseline responses were measured in PBS-T. Biosensors were loaded with biotinylated fentanyl-hapten (where the biotin replaces the poly-gly sequence from fen-G4; biotinylated fentanyl derivative syntheses are described in^{33 (link)}), 0.2 μg/mL for 60 s. Association of antibody with biotinylated hapten was measured in PBS-T with 5 sample concentrations ranging from 5 nM–200 nM for 180 s, followed by dissociation in PBS-T for 300 s. Isothermal titration calorimetry (ITC) experiments were performed using Microcal PEAQ-ITC (Malvern, UK). For all experiments, PBS was used to dissolve fentanyl-citrate (Sigma, 2 mg/mL), fentanyl-hapten, and for the final purification step of Fabs. Titrations were performed at 25°C by injecting consecutive (1–3 μL) aliquots of fentanyl or fentanyl-hapten (both 50 μM) into Fab fragment (4–7 μM) with 120 s intervals. The binding data was corrected for the heat of dilution and fit to a one-site binding model to calculate the K_d, and the binding parameters, N and ΔH. Binding sites were assumed to be identical.

Rajantie I., Ekman N., Iljin K., Arighi E., Gunji Y., Kaukonen J., Palotie A., Dewerchin M., Carmeliet P, & Alitalo K. (2001). Bmx Tyrosine Kinase Has a Redundant Function Downstream of Angiopoietin and Vascular Endothelial Growth Factor Receptors in Arterial Endothelium. Molecular and Cellular Biology, 21(14), 4647-4655.

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Kinetic Analysis of Miro1-Nbs Binding

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Analysis of binding kinetics of Miro1-specific Nbs was performed using the Octet RED96e system (Sartorius) according to the manufacturer's recommendations. In brief, 2 -10 µg/mL solution of biotinylated Miro1-Nbs diluted in Octet buffer (HEPES, 0.1% BSA) was used for 40 s to immobilize the Nb on streptavidin coated biosensor tips (SA, Sartorius). In the association step, a dilution series of Miro1 ranging from 3.9 nM -1 µM were reacted for 300 s followed by dissociation in Octet buffer for 720 s. Every run was normalized to a reference run using Octet buffer for association. Data were analyzed using the Octet Data Analysis HT 12.0 software applying the 1:1 ligand-binding model and global fitting.

Fagbadebo F.O., Kaiser P.D., Zittlau K., Bartlick N., Wagner T.R., Froehlich T., Jarjour G., Nueske S., Scholz A., Traenkle B., Maček B., & Rothbauer U. (2021). A nanobody-based toolset to monitor and modify the mitochondrial GTPase Miro1.

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