Streptavidin coated biosensors

Manufactured by Molecular Devices

Sourced in United States

Streptavidin-coated biosensors are a type of lab equipment designed to detect and measure the presence of specific biomolecules in a sample. The core function of these biosensors is to utilize the high-affinity binding between streptavidin and biotin, a widely used biochemical tool, to capture and detect target analytes.

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30 protocols using streptavidin coated biosensors

Octet RED96 System Protein-Protein Interactions

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Protein–protein interactions were measured by using an Octet RED96 System (ForteBio) using streptavidin-coated biosensors (ForteBio). Each well contained 200 μL of solution, and the assay buffer was HBS-EP+ buffer (GE Healthcare Life Sciences, 10 mM HEPES pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.05% (v/v) surfactant P20) plus 0.5% non-fat dry milk blotting grade blocker (BioRad). The biosensor tips were loaded with analyte peptide or protein at 20 μg mL⁻¹ for 300 s (threshold of 0.8 nm response), incubated in HBS-EP+ buffer for 60 s to acquire the baseline measurement, dipped into the solution containing cage and/or key for 1800 s (association step) and dipped into the HBS-EP+ buffer for 1800 s (dissociation steps). The binding data were analysed with the ForteBio Data Analysis Software version 9.0.0.10.

Zhang J.Z., Yeh H.W., Walls A.C., Wicky B.I., Sprouse K.R., VanBlargan L.A., Treger R., Quijano-Rubio A., Pham M.N., Kraft J.C., Haydon I.C., Yang W., DeWitt M., Bowen J.E., Chow C.M., Carter L., Ravichandran R., Wener M.H., Stewart L., Veesler D., Diamond M.S., Greninger A.L., Koelle D.M, & Baker D. (2022). Thermodynamically coupled biosensors for detecting neutralizing antibodies against SARS-CoV-2 variants. Nature Biotechnology, 40(9), 1336-1340.

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Quantifying Fortilin-PRX1 Interaction

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Recombinant fortilin protein produced as described above was biotinylated and
immobilized on streptavidin-coated biosensors (ForteBio, Menlo Park, CA) at a
concentration of 1 μg/mL in BI Buffer (25 mM
Tris, 150 mM NaCl, 0.1% Tween-20) for 600 seconds,
followed by buffer exchange into PBS. We then added various concentrations of
recombinant PRX1 (Sigma-Aldrich, 0 to 5000 μM) for
180 seconds to evaluate the association between the two molecules.
Finally, we replaced the solution with PBS for 300 seconds to
evaluate their dissociation. The binding data were processed and a dissociation
constant was calculated by using BLItz analysis software (Forte Bio).

Chattopadhyay A., Pinkaew D., Doan H.Q., Jacob R.B., Verma S.K., Friedman H., Peterson A.C., Kuyumcu-Martinez M.N., McDougal O.M, & Fujise K. (2016). Fortilin potentiates the peroxidase activity of Peroxiredoxin-1 and protects against alcohol-induced liver damage in mice. Scientific Reports, 6, 18701.

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Binding Kinetics of pHis Fabs

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The binding kinetics of pHis Fabs with the peptides (pTza and pHis) were evaluated with an Octet RED96e (FortéBio). Streptavidin-coated biosensors (FortéBio) were presoaked in buffer containing 50 mM Hepes pH 8.0, 150 mM NaCl, 0.05% Tween-20, and 0.2% BSA for 15 min. The biotinylated pTza peptides or pHis peptides (20 nM) with and without phosphoramidate treatment were coated onto these streptavidin biosensors and analyzed for their binding to varying concentrations of the pHis Fabs with an association time of 120 s and dissociation time of 200 s. The entire set of experiments was repeated three times. The data were analyzed using the Octet software (FortéBio) and the rates of association and dissociation were obtained by global fitting data to the 1:1 Langmuir interaction model.

Kalagiri R., Stanfield R.L., Meisenhelder J., La Clair J.J., Fuhs S.R., Wilson I.A, & Hunter T. (2021). Structural basis for differential recognition of phosphohistidine-containing peptides by 1-pHis and 3-pHis monoclonal antibodies. Proceedings of the National Academy of Sciences of the United States of America, 118(6), e2010644118.

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Quantifying Protein-Protein Interactions

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Protein–protein interactions were measured by using an Octet RED96 System (ForteBio) using streptavidin-coated biosensors (ForteBio). Each well contained 200 μl solution, and the assay buffer was HBS-EP + buffer (GE Healthcare Life Sciences, 10 mM HEPES, pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.05% (v/v) surfactant P20) plus 0.5% non-fat dry milk blotting grade blocker (Bio-Rad). The biosensor tips were loaded with analyte peptide or protein at 20 μg ml⁻¹ for 300 s (threshold of 0.8-nm response), incubated in HBS-EP + buffer for 60 s to acquire the baseline measurement, dipped into the solution containing cage and/or key for 1,800 s (association step) and dipped into the HBS-EP + buffer for 1,800 s (dissociation steps). The binding data were analyzed with the ForteBio Data Analysis Software version 9.0.0.10.

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Binding Affinity Measurement of RavA and LdcI Proteins

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For BLI binding studies, RavA with a biotinylated C-terminal AviTag was expressed and purified as previously described (28 (link)). BLI experiments were performed in 1× Hepes buffered saline (HBS) with a pH of 7.0 (25 mM Hepes, 300 mM NaCl, 10 mM MgCl₂, 10% glycerol) supplemented with 1× kinetics buffer (0.1% wt/vol BSA, 0.02% vol/vol Tween-20), 1 mM ADP, 1 mM DTT, and 0.1 mM PLP. Experiments were performed using the BLItz System instrument (FortéBio), operated at room temperature. Before the start of each BLI experiment, RavA-AviTag was incubated with 1 mM ADP for 10 min. Streptavidin-coated biosensors (FortéBio) were functionalized with biotinylated RavA-AviTag, then quenched with 10 μg/mL biocytin. Experiments with the wild-type LdcI are adapted from ref. 28 (link). For C-terminal LdcI–FP fusions, pins were dipped in wells containing a range of LdcI–FP concentrations from 0 to 1,000 nM, with no binding signal recorded at any concentration of LdcI–FP.

Jessop M., Liesche C., Felix J., Desfosses A., Baulard M., Adam V., Fraudeau A., Huard K., Effantin G., Kleman J.P., Bacia-Verloop M., Bourgeois D, & Gutsche I. (2020). Supramolecular assembly of the Escherichia coli LdcI upon acid stress. Proceedings of the National Academy of Sciences of the United States of America, 118(2), e2014383118.

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Virus-Sialylated Receptor Binding Assay

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Purified virus binding to different sialylated receptor analogues was tested using an Octet RED biolayer interferometer (Pall ForteBio), as described previously (18 (link)). Receptor analogues contained 30-kDa polyacrylamide backbones conjugated to 20 mol% trisaccharides and 5 mol% biotin (Lectinity Holdings). The three analogues used in this study were α2,6-sialyllactosamine (6SLN), α-2,3-sialyllactosamine (3SLN), and Neu5Ac α-2,3Galβ1-4(6-HSO3)GlcNAc (3SLN[6su]). Sialoglycopolyemers were bound onto streptavidin-coated biosensors (Pall ForteBio) at ranges of concentrations from 0.01 to 0.55 μg/ml in HBS-EP (10 mM HEPES [pH 7.4], 150 mM NaCl, 3 mM EDTA, 0.005% Tween 20). Virus was diluted to a concentration of 100 pM in HBS-EP, 10 μM oseltamavir carboxylate (Roche), and 10 μM zanamivir (GSK). Virus association to the bound sialoglcopolymers was measured at 20°C for 30 min. Virus binding curves were normalized to fractional saturation and plotted as a function of sugar loading. Relative dissociation constants were calculated as described previously (18 (link), 25 (link)).

Peacock T.P., Sealy J.E., Harvey W.T., Benton D.J., Reeve R, & Iqbal M. (2021). Genetic Determinants of Receptor-Binding Preference and Zoonotic Potential of H9N2 Avian Influenza Viruses. Journal of Virology, 95(5), e01651-20.

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Epitope Binning of Anti-PvDBPII Monoclonal Antibodies

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BLI was carried out on an OctetRED384 (Pall FortéBio) using streptavidin-coated biosensors (Pall FortéBio) to immobilise PvDBPII enzymatically monobiotinylated on a C-terminal AviTag™. Assays were carried out in 96-well format in black plates (Greiner). For epitope binning studies (Figure 1E-G), a six-step sequential assay was performed: Baseline (PBS, 30 s); Protein immobilisation (neat supernatant, 120 s); Wash (PBS, 60 s); first mAb (mAb1) binding (300 nM mAb1, 120 s); Wash (PBS, 60 s); second mAb (mAb2) binding (150 nM mAb2, 120 s). “Relative binding” in Figure 1E shows the ratio (Signal_mAb2 with mAb1 bound)/(Signal_mAb2 with no mAb1) where “Signal_mAb2” was normalised for the amount of PvDBPII bound to the biosensor, such that “Signal_mAb2” = the raw signal in “mAb2 binding” divided by the raw signal in the “Protein immobilisation” phase. The resulting “binding profile” for any given mAb corresponds to the column of “relative binding values” under that mAb in the “relative binding” table. To establish the epitope bins, binding profiles between each mAb pair were correlated using a Pearson product-moment correlation coefficient, the values of which are shown in the “binding profile correlation” matrix in Figure 1F. mAb pairs whose binding profile correlation was > 0.7 were grouped into the same epitope bin (Figure 1G).

Rawlinson T.A., Barber N.M., Mohring F., Cho J.S., Kosaisavee V., Gérard S.F., Alanine D.G., Labbé G.M., Elias S.C., Silk S.E., Quinkert D., Jin J., Marshall J.M., Payne R.O., Minassian A.M., Russell B., Rénia L., Nosten F.H., Moon R.W., Higgins M.K, & Draper S.J. (2019). Structural basis for inhibition of Plasmodium vivax invasion by a broadly neutralising vaccine-induced human antibody. Nature microbiology, 4(9), 1497-1507.

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Kinetic Evaluation of AlbiVax-Albumin Binding

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The kinetic binding of AlbiVax with albumin was measured by BLI on an Octet Red96 system (fortéBio) using streptavidin-coated biosensors (fortéBio). Assays were carried out with agitation in solid black 96-well plates (Geiger Bio-One) in seven steps: 1, baseline (1x PBS), time: 60 s; 2, loading (Biotin-labeled albumin, 1 µg mL⁻¹), time: 600 s; 3, baseline (1× PBS), time: 60 s; 4, quenching (1 µg mL⁻¹ Biocytin (Thermo Scientific)), time: 180 s; 5, baseline (1× PBS), time: 60 s; 6, association, time: 600 s; 7, dissociation, time: 600 s. Nonspecific binding was performed by measuring the binding of albumin-loaded biosensor to buffer alone and blank biosensor to analytes. Data analysis and curve fitting were performed using Octet Analysis software 7.0. Binding data were fitted with a binding model of 1:1 ligand interaction using GraphPad Prism 7 (La Jolla, CA).

Zhu G., Lynn G.M., Jacobson O., Chen K., Liu Y., Zhang H., Ma Y., Zhang F., Tian R., Ni Q., Cheng S., Wang Z., Lu N., Yung B.C., Wang Z., Lang L., Fu X., Jin A., Weiss I.D., Vishwasrao H., Niu G., Shroff H., Klinman D.M., Seder R.A, & Chen X. (2017). Albumin/vaccine nanocomplexes that assemble in vivo for combination cancer immunotherapy. Nature Communications, 8, 1954.

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Measuring Protein Binding Affinity

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The binding affinity for the minibinders were determined by the Octet RED96 (ForteBio). To measure the binding affinity, streptavidin-coated biosensors (ForteBio) were first loaded with biotinylated target proteins at 50~100nM concentration, washed with Octet buffer (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.05% surfactant P20 and 1% BSA), and incubated with titrated concentrations of corresponding binders. To measure the Koff, the biosensors were then dipped back into the Octet buffer. The Kon, Koff and KD were further estimated with the Octet Analysis software.

Huang B., Abedi M., Ahn G., Coventry B., Sappington I., Wang R., Schlichthaerle T., Zhang J.Z., Wang Y., Goreshnik I., Chiu C.W., Chazin-Gray A., Chan S., Gerben S., Murray A., Wang S., O’Neill J., Yeh R., Misquith A., Wolf A., Tomasovic L.M., Piraner D.I., Gonzalez M.J., Bennett N.R., Venkatesh P., Satoe D., Ahlrichs M., Dobbins C., Yang W., Wang X., Vafeados D., Mout R., Shivaei S., Cao L., Carter L., Stewart L., Spangler J.B., Bernardes G.J., Roybal K.T., Greisen P J.r., Li X., Bertozzi C, & Baker D. (2023). Designed Endocytosis-Triggering Proteins mediate Targeted Degradation. bioRxiv.

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BLI Assay of MSI2-RNA Binding

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ForteBio Octet RED 96 (Forte Bio, Fremont, CA, USA) was used to perform the BLI assay. Briefly, streptavidin-coated biosensors (Forte Bio) were bound to the biotinylated RNA oligonucleotides and incubated with protein samples at different concentrations to measure the binding kinetics between the MSI2 protein and biotinylated RNA oligonucleotides. The detailed procedure was described in the Supplemental Methods.

Guo Q.Q., Gao J., Wang X.W., Yin X.L., Zhang S.C., Li X., Chi L.L., Zhou X.M., Wang Z, & Zhang Q.Y. (2021). RNA-Binding Protein MSI2 Binds to miR-301a-3p and Facilitates Its Distribution in Mitochondria of Endothelial Cells. Frontiers in Molecular Biosciences, 7, 609828.

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