Purified proteins were diluted or dialyzed in dialysis buffer (KH2PO4 42 mM, Na2HPO4 8 mM, NaCl 136 mM, KCl 2.6 mM, pH 7.4, Tween 20 0.02%) and biotinylated using the EZ-Link®NHS-PEO4-Biotinylation Kit (Thermo Fisher Scientific, Waltham, MA, USA) for 30 min at room temperature. The unconjugated biotin was removed by Zeba Spin desalting column (Thermo Fisher Scientific, Waltham, MA, USA). The biotin-conjugated protein was diluted to 50 µg/mL. The Super Streptavidin sensors (Fortebio, Silicon Valley, CA, USA) were pre-wetted in dialysis buffer for 15 min prior to use and then loaded with biotinylated proteins for 15 min. The sensors unloaded with biotinylated proteins or loaded with rTrx was used as controls to correct for base-line drift. Progesterone was prepared in serial dilutions (0.125, 0.25, 0.5, 1, 2, and 4 mM) in a 96-well plate. Measurements were carried out automatically at room temperature using the Fortebio’s Octet System and analyzed by Octet System Data Analysis. Every experiment was repeated twice.
Octet system
Manufactured by Molecular Devices
Sourced in United States
The Octet system is a label-free, real-time biomolecular interaction analysis platform. It enables the measurement of kinetics, affinity, and concentration of biomolecular interactions between proteins, peptides, small molecules, and other analytes.
Automatically generated - may contain errors
Lab products found in correlation
Octet data analysis software 8, by Molecular Devices (4 mentions)
Streptavidin biosensor, by Molecular Devices (3 mentions)
Anti gst biosensors, by Molecular Devices (2 mentions)
Centricon centrifugal filter with a mwco of 10 000, by Merck Group (2 mentions)
Ez link nhs peg4 biotin, by Thermo Fisher Scientific (2 mentions)
Bovine serum albumin powder, by Merck Group (2 mentions)
Octet nta, by Molecular Devices (2 mentions)
Ht v10, by Molecular Devices (2 mentions)
Octet fortebio software, by Molecular Devices (2 mentions)
Kinetic buffer, by Molecular Devices (2 mentions)
Ez link nhs peo4 biotinylation kit, by Thermo Fisher Scientific (1 mentions)
Zeba spin desalting column, by Thermo Fisher Scientific (1 mentions)
Super streptavidin sensors, by Molecular Devices (1 mentions)
Dr5 fc, by R&D Systems (1 mentions)
Octet analysis software, by Molecular Devices (1 mentions)
Arg2 tips, by Molecular Devices (1 mentions)
Nta biosensor, by Molecular Devices (1 mentions)
Ct009 h08h, by Sino Biological (1 mentions)
Lipopolysaccharides (lps), by Merck Group (1 mentions)
Originpro 2015, by OriginLab (1 mentions)
37 protocols using octet system
1
Biotin-Conjugation and Progesterone Binding Assay
2
Binding Affinity Measurements of Anti-BTLA Antibodies
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Example 4
Binding affinities of anti-BTLA antibodies to human and cynomolgus BTLA were measured by Bio-Layer Interferometry (Octet® systems from FortéBIO). 10 μg/ml of each anti-BTLA antibody was immobilized on the biosensor tip surface via anti-human-Fc (AHC) capture. Human or cynomolgus BTLA was diluted in PBS and loaded at concentrations from 166.7 nM to 6.17 nM in 3-fold serial dilutions. Binding curves were fitted to a 1:1 interaction model using the analysis software provided by the Octet® systems.
3
Measuring TRAIL-Death Receptor Binding
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Biolayer interferometry performed on Octet systems (Pall Forte Bio LLC, CA) was used to measure the affinity of TRAIL proteins for death receptors. The death receptor proteins DR4-Fc and DR5-Fc (Sino Biological, Beijing, China) were immobilized on a protein A-coated biosensor followed by insertion into solutions containing different concentrations of TRAIL proteins for association according to our previous works (Yang et al., 2018 (link)). In addition, death receptor binding was also measured by neutralizing the cytotoxicity of TRAIL proteins. Briefly, in the cytotoxicity assay system, TRAIL proteins (2.5–50 nM) were mixed with DR4-Fc or DR5-Fc at different molar ratios (DR protein: TRAIL = 0–3) prior to addition to cells. The viabilities of cells treated with TRAIL proteins in the presence or absence of death receptor proteins were measured and compared. The increase in the viability of cancer cells after treatment with TRAIL proteins in the presence of death receptor proteins reflects the binding of TRAIL proteins to death receptors.
4
Biolayer Interferometry Analysis of Protein Interactions
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Biolayer interferometry performed on Octet® systems (Pall ForteBio LLC, CA) was used to reveal protein-protein interactions. For IgG binding assay, IgG was immobilized on a protein A-coated sensor. Subsequently, the sensor was inserted into a solution containing different concentrations (0-2 μM) of IgBD-TRAIL for 300 s to enable association, followed by disassociation in PBS for 200 s. The association constant ka, disassociation constant kd, and affinity KD (KD=kd/ka) were calculated by using software for Octet® systems according to 1:1 binding model. For death receptor binding assay, DR4-Fc or DR5-Fc (R&D, MN) were captured by a protein A-coated sensor, followed by the insertion of the sensor into solutions containing different concentrations of TRAIL fusion proteins. For albumin binding assays, ABD-TRAIL was captured by using a His-tag antibody-coated sensor, followed by the insertion of the sensor into a series of albumin solutions.
5
Lectin-based Glycoprotein Quantification
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Relative quantification of N-glycans present on the target-binding domain was performed by lectin-based bio-layer interferometry using OctetSystems (ForteBio). Before the lectin assay, Fc-fusion protein concentrations were determined as described above, followed by sam-
6
Affinity Characterization of Humanized Nb1902-Fc
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Example 7
(1) The humanized single domain antibody Nb1902-Fc was gradient diluted from 100 nM with PBST, respectively: 100 nM, 66.7 nM, 44.4 nM, 29.6 nM, 19.8 nM, 13.2 nM. The antigen proteins hCD47 (ECD)-Fc and Fc were diluted to 30 μg/mL, respectively. (2) Setting the operating conditions of the instrument: temperature 30° C., shake speed 1000 rpm. Using ProteinA-coated probe (Fortebio Part No: 18-5010) to capture antibody for 180s; binding the gradient diluted antigen for 180s; 300s of dissociation time; 10 mM glycine (PH1.7) regeneration 3 times, 5s for each time. (3) Using ForteBio's Octet System for on-board testing.
The test results are shown in
7
Measuring Antibody-Antigen Interactions
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Bio-layer interferometry measurements were performed using anti–human IgG sensors in an Octet system (ForteBio). Anti-αIIbβ3 mAbs (10 μg/mL) were captured on the sensors for 10 minutes. Equilibrium dissociation constants (KD) were determined by monitoring over 85 minutes the association between the immobilized antibodies and αIIbβ3 in solution in duplicate for 7 concentrations of antigen (200, 100, 50, 25, 12.5, 6.25, and 3.13 nM). An irrelevant IgG mAb was used as negative control to subtract the background signal. Data analysis was performed using the Octet Analysis software (ForteBio), and KD values were calculated by steady-state analysis.
8
Binding Kinetics of Anti-CD71 Fab
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Example 23
This Example shows that an anti-human CD71 antigen-binding fragment (Fab) of the present disclosure, which is derived from anti-human CD71 antibody of the present disclosure, demonstrated a binding affinity to recombinant human and cynomolgus CD71 protein.
A Fab antigen-binding fragment was generated by digestion of anti-CD71 21.12 antibody of the present disclosure with the papain enzyme in accordance with known protocols to generate an anti-CD71 Fab fragment. The anti-CD71 Fab fragment of the present disclosure was assayed for binding to recombinant human or cynomolgus CD71 protein by measurement of the kinetic on- and off-rates of a 1:3 dilution series of the Fab fragment to a substrate-immobilized recombinant CD71 protein (Octet system, ForteBio). The recombinant CD71 proteins included a hexa-histidine (His6) peptide tag, by which the protein was immobilized to a Ni-NTA (nitrilotriacetic acid)-containing substrate. The results are shown in Table 19.
9
Binding Affinity Analysis of SARS-CoV-2 Antibodies
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Binding studies were carried out using the Octet system (ForteBio, USA, Version 8.1, 2015) that measures biolayer interferometry (BLI) as previously described [18 (link)]. All steps were performed at 30 °C with shaking at 1500 rpm in a black 96-well plate containing 200 μl solution per well. For affinity measurements, streptavidin-coated biosensors were loaded with biotinylated antibodies (5 μg/ml) to reach 1-nm wavelength shift followed by a wash. The sensors were then reacted for 300 s with increasing concentrations of RBD/NTD (association phase) and then transferred to buffer-containing wells for another 600 s (dissociation phase). Binding and dissociation were measured as changes over time in light interference after subtraction of parallel measurements from unloaded biosensors. Sensorgrams were fitted with a 1:1 binding model using the Octet data analysis software 8.1 (Fortebio, USA, 2015), and the presented values are an average of several repeated measurements (at least two repeats). For binning experiments of antibody pairs, antibody-loaded sensors were incubated with a fixed S1 concentration (200 nM), washed and incubated with the non-labeled antibody counterpart. For concomitant binding, antibodies were consecutively loaded on the pre-existing antibody-antigen complex, with no regeneration step.
10
DltB-DltC Binding Interaction Assay
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