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Molecular Devices' Data Analysis software provides a comprehensive platform for analyzing and visualizing experimental data. The software offers a range of tools for data processing, statistical analysis, and graphical representation, enabling researchers to efficiently manage and interpret their research findings.

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81 protocols using data analysis software

Kinetics of Small Molecule Binding to KK-LC-1

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The binding affinity of the small molecule to KK-LC-1 protein was determined by BLI using a Forte Bio Octet K2 system. All assays were run at 30 °C with continuous shaking at 1000 rpm. The assay buffer consisted of PBS with 0.1% BSA, 0.01% Tween-20, and 1% DMSO. 0.15 mg/ml KK-LC-1 protein in sterile water was biotinylated and immobilized on Super Streptavidin (SSA) biosensors. Small molecule drugs were dissolved in PBS and adjusted to different concentrations. Sensors were washed with assay buffer for 10 min after each round of association and disassociation to remove nonspecifically bound protein and establish a baseline. Raw kinetic data were generated using Data Acquisition software (ForteBio). Association/dissociation rate constants (k_on/k_off) were generated with the double reference subtraction method using Data Analysis software (ForteBio), and affinities (KD) were calculated.

Bu J., Zhang Y., Wu S., Li H., Sun L., Liu Y., Zhu X., Qiao X., Ma Q., Liu C., Niu N., Xue J., Chen G., Yang Y, & Liu C. (2023). KK-LC-1 as a therapeutic target to eliminate ALDH+ stem cells in triple negative breast cancer. Nature Communications, 14, 2602.

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Biolayer Interferometry Antibody Binding

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Competition-binding studies were performed using a biolayer interferometry instrument (FortéBio Octet HTX). HIS1K sensortips were preincubated in kinetic buffer (Pall) for 10 min. After a 60-s baseline step, His-tagged YFV E protein (Meridian Life Science) was associated with the tips at 5 μg/mL for 60 s. Readings were again set to the baseline for 60 s, followed by the association of the first antibody at 25 μg/mL for 600 s to achieve complete saturation. Tip readings were again set to the baseline, and the tips were then dipped into wells containing a second antibody at 25 μg/mL for 180 s. Data were analyzed using FortéBio data analysis software. Data from all steps were normalized to a buffer-only control, and antibodies were grouped using Pearson correlation statistical analysis.

Doyle M.P., Genualdi J.R., Bailey A.L., Kose N., Gainza C., Rodriguez J., Reeder K.M., Nelson C.A., Jethva P.N., Sutton R.E., Bombardi R.G., Gross M.L., Julander J.G., Fremont D.H., Diamond M.S, & Crowe JE J.r. (2022). Isolation of a Potently Neutralizing and Protective Human Monoclonal Antibody Targeting Yellow Fever Virus. mBio, 13(3), e00512-22.

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Kinetic Analysis of 48.1-scFv-Fc Binding

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Binding kinetics of 48.1-scFv-Fc was performed using Octet RED (Fortebio, USA) with anti-human Fc sensors. Kinetic cycle consisted of: 1) Equilibrium—sensors were dipped in wells containing kinetic buffer (0.02%/tween, 0.1%/BSA, PBS) for 60 sec. 2) Loading—sensors were dipped in wells containing supernatant of cells transfected with 48.1 (Supplementary Fig. 5c) or 0.05 μg/ml of purified 48.1 scFv for 300 sec (Supplementary Fig. 5d). 3) Baseline—sensors were dipped into wells containing kinetic buffer for 180 sec. 4) Association—sensors were dipped into wells containing 10 μg/ml HA proteins H1CA0709, H2JPN57 (H2 A/Japan/305/1957, IRR FR-700), H5VN04 (protein sciences), H3E09 and H7N3 (H7 A/Netherlands/219/2003, IRR FR-71) (Supplementary Fig. 5c) or serial dilutions of H2JPN57 (Supplementary Fig. 5d) for 300 sec. 5) Dissociation—sensors were dipped into wells containing kinetic buffer for 300 sec (Supplementary Fig. 5c) or 1200 sec (Supplementary Fig. 5d). Association rate (Kon) dissociation rate (kdis) and equilibrium dissociation (KD) constants were calculated using 1:1 fitting model as provided by the Fortebio Data analysis software.

Avnir Y., Watson C.T., Glanville J., Peterson E.C., Tallarico A.S., Bennett A.S., Qin K., Fu Y., Huang C.Y., Beigel J.H., Breden F., Zhu Q, & Marasco W.A. (2016). IGHV1-69 polymorphism modulates anti-influenza antibody repertoires, correlates with IGHV utilization shifts and varies by ethnicity. Scientific Reports, 6, 20842.

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Affinity Measurement and Epitope Binning of Anti-LILRB3 Antibodies

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Affinity measurement and epitope binning were done as described previously^{19 (link)}. Briefly, antibody affinity was analyzed with the Octet RED96 instrument. Antibody (30 mg/mL) was loaded onto the protein A biosensors then exposed to a series of concentrations of recombinant LILRB3 (0.1–200 nmol/L), and background subtraction was used to correct for sensor drift. ForteBio’s data analysis software was used to extract association and dissociation rates assuming a 1:1 binding model. The K_d was calculated as the ratio k_off/k_on. Epitope binning of anti-LILRB3 rabbit antibodies was performed with an Octet RED96 instrument using a classical sandwich epitope binning assay. In these epitope binning assays, primary antibodies (40 μg/ml) were loaded onto protein A biosensors, and remaining Fc-binding sites on the sensor were blocked with a human non-targeting IgG (200 μg/ml). The sensors were then exposed to the 1 μM LILRB3 diluted in 1× kinetics buffer, followed by the secondary antibodies (40 μg/ml). Raw data were processed using ForteBio’s data analysis software 7.0. Antibody pairs were assessed for competitive binding. Additional binding by the secondary antibody indicates an unoccupied epitope (the antibodies of the pair are not competitors), and no binding indicates epitope blocking (the antibodies of the pair are competitors for the same epitope).

Wu G., Xu Y., Schultz R.D., Chen H., Xie J., Deng M., Liu X., Gui X., John S., Lu Z., Arase H., Zhang N., An Z, & Zhang C.C. (2021). LILRB3 supports acute myeloid leukemia development and regulates T-cell antitumor immune responses through the TRAF2–cFLIP–NF-κB signaling axis. Nature cancer, 2(11), 1170-1184.

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Kinetic Analysis of Anti-IFN-γ mAbs

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Kinetics of mouse anti-IFN-γ mAbs (B27, B133.5, and MD-1) were analyzed using a ForteBio Octet RED96e instrument. All the assays were performed at 200 μl/well in 0.05% Tween 20-PBS at 30°C. Ten micrograms per milliliter of His-tagged IFN-γ was loaded onto the surface of anti-penta His biosensors (HIS1K), followed by a 120 s biosensor washing step. The association of IFN-γ on the biosensor to the individual mAbs in solution was analyzed for 120 s. The dissociation of the interaction was probed for 600 s. The K_D was calculated using the ratio k_d/k_a.
For epitope binning, His-tagged IFN-γ was captured onto anti-penta His biosensors and was coated with testing mAbs (antibody bin) at a saturating concentration. The epitopes of the other mAbs were probed in relation to the testing mAbs. Raw data were processed using the ForteBio Data Analysis Software, and the antibody pairs were assessed for competitive binding. Additional binding by the second antibody indicated an unoccupied epitope (non-competitor), while no binding indicated epitope blocking (competitor).

Yasamut U., Thongkum W., Moonmuang S., Sakkhachornphop S., Chaiwarith R., Praparattanapan J., Wipasa J., Chawansuntati K., Supparatpinyo K., Lai E, & Tayapiwatana C. (2019). Neutralizing Activity of Anti-interferon-γ Autoantibodies in Adult-Onset Immunodeficiency Is Associated With Their Binding Domains. Frontiers in Immunology, 10, 1905.

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Antibody Affinity Measurement Using BLI

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For antibody affinity measurement, antibody (30 μg/mL) was loaded onto the protein G biosensors for 4 minutes. Following a short baseline in kinetics buffer, the loaded biosensors were exposed to a series of recombinant LILRB4 concentrations (0.1–200 nM) and background subtraction was used to correct for sensor drifting. All experiments were performed with shaking at 1,000 rpm. Background wavelength shifts were measured from reference biosensors that were loaded only with antibody. ForteBio’s data analysis software was used to fit the data to a 1:1 binding model to extract an association rate and dissociation rate. The K_d was calculated using the ratio k_off/k_on.

Gui X., Deng M., Song H., Chen Y., Xie J., Li Z., He L., Huang F., Xu Y., Anami Y., Yu H., Yu C., Li L., Yuan Z., Xu X., Wang Q., Chai Y., Huang T., Shi Y., Tsuchikama K., Liao X.C., Xia N., Gao G.F., Zhang N., Zhang C.C, & An Z. (2019). Disrupting LILRB4/APOE interaction by an efficacious humanized antibody reverses T-cell suppression and blocks AML development. Cancer immunology research, 7(8), 1244-1257.

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Kinetic Analysis of Fab-Peptide Binding

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Binding kinetics of mAb41 Fab to selected peptides were measured using a Fortebio Octet Red 96 instrument (Menlo Park, CA). All assays were performed in 1 × kinetic buffer (Fortebio). Biotinylated peptides were loaded onto streptavidin biosensors (Fortebio) for 100 s and quenched with biocytin. A two-fold dilution series of antibody Fab (prepared using a mouse IgG1 kit, Pierce) was used as analyte. Association was performed for 300 s and dissociation for 900 s. Binding constants were obtained by fitting sensorgrams with a 1:1 model using ForteBio Data Analysis Software.

Guo N., Duan H., Kachko A., Krause B.W., Major M.E, & Krause P.R. (2015). Reverse Engineering of Vaccine Antigens Using High Throughput Sequencing-enhanced mRNA Display. EBioMedicine, 2(8), 859-867.

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Monoclonal Antibody Binding Kinetics to SARS-CoV-2 Spike

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Octet K2 system (ForteBio) was used to determine the monoclonal antibody binding with S-proteins or selected peptides. IgG was first captured for 60s by anti-human IgG Fc capture (AHC) biosensors (ForteBio Cat# 18-5063), then baseline was provided in Octet buffer (PBS with 0.1% Tween) for another 60s. After that, the sensors were transferred into wells containing diluted HCoV S-proteins for 120s for association, and into Octet buffer for disassociation for 240s. Selected peptides that were N-terminal biotinylated were diluted in Octet buffer and first captured for 60s by the hydrated streptavidin biosensors (ForteBio Cat# 18-5020), then unbound peptides were removed by transferring into Octet buffer for 60s to provide the baseline. The sensors were then immersed into the monoclonal antibodies in Octet buffer for 120s for association, followed by transferring into Octet buffer for 240s for dissociation. The data generated were analyzed using the ForteBio Data Analysis software for correction, and the kinetic curves were fit to a 1:1 binding mode. Note that the IgG: spike protomer binding can be a mixed population of 2:1 and 1:1, such that the term ‘apparent affinity’ dissociation constants (K_D^App) are shown to reflect the binding affinity between IgGs and spike trimers tested.

Zhou P., Song G., Liu H., Yuan M., He W.T., Beutler N., Zhu X., Tse L.V., Martinez D.R., Schäfer A., Anzanello F., Yong P., Peng L., Dueker K., Musharrafieh R., Callaghan S., Capozzola T., Limbo O., Parren M., Garcia E., Rawlings S.A., Smith D.M., Nemazee D., Jardine J.G., Safonova Y., Briney B., Rogers T.F., Wilson I.A., Baric R.S., Gralinski L.E., Burton D.R, & Andrabi R. (2023). Broadly neutralizing anti-S2 antibodies protect against all three human betacoronaviruses that cause deadly disease. Immunity, 56(3), 669-686.e7.

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Biolayer Interferometry Analysis of HSF2 Interactions

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For in vitro protein-protein interaction experiments, we used biolayer interferometry technology (Octet Red, Forté-Bio, USA). Recombinant HSF2 (TP310751 Origen) was desalted (ZebaTM Spin Desalting Columns, 7 K molecular weight cutoff, 0.5 ml (1034–1164, Fisher Scientific, Germany)) and biotinylated at a molar ratio biotin/protein (3:1) for 30 min at room temperature (EZ-Link NHS-PEG4-Biotin [1189–1195, Fisher Scientific, Germany]). Excess Biotin was removed using ZebaTM Spin Desalting Columns. Biotinylated recombinant HSF2 was used as a ligand and immobilized at 100 nM on streptavidin biosensors after dilution in phosphate-buffered saline (PBS; 600 s). Interactions with desalted analytes diluted in PBS at 100 nM (recombinant CBP domains 6 His-tag Full-HAT, Bromodomain [BD], RING, or HSP70 as a positive control [ADI-SPP-555, Enzo-Life Sciences]) were analyzed after association (600 s). All sensorgrams were corrected for baseline drift by subtracting a control sensor exposed to running buffer only. For Kd determination, each Kd was calculated with a 1:1 stoichiometry model using a global fit with Rmax unlinked by sensor (FortéBio, Data analysis software version 7.1.0.89).

de Thonel A., Ahlskog J.K., Daupin K., Dubreuil V., Berthelet J., Chaput C., Pires G., Leonetti C., Abane R., Barris L.C., Leray I., Aalto A.L., Naceri S., Cordonnier M., Benasolo C., Sanial M., Duchateau A., Vihervaara A., Puustinen M.C., Miozzo F., Fergelot P., Lebigot É., Verloes A., Gressens P., Lacombe D., Gobbo J., Garrido C., Westerheide S.D., David L., Petitjean M., Taboureau O., Rodrigues-Lima F., Passemard S., Sabéran-Djoneidi D., Nguyen L., Lancaster M., Sistonen L, & Mezger V. (2022). CBP-HSF2 structural and functional interplay in Rubinstein-Taybi neurodevelopmental disorder. Nature Communications, 13, 7002.

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Binding Kinetics of rEF1a with Igl-C Protein

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Binding kinetics of rEF1a with Igl-C protein or an unrelated protein (bovine serum albumin, BSA) were analyzed by biolayer interferometry using an Octet-Red96 device (Pall ForteBio, Menlo Park, CA, USA). Purified Igl-C and BSA in 30 μg/mL solutions of buffered sodium acetate (pH 4.0) and sodium acetate (pH 5.0), respectively, were immobilized onto activated AR2G biosensors until saturation. Twofold serial dilutions of rEF1a protein were prepared from 4000 nM to 250 nM. AR2G biosensors were then incubated with the serial dilutions in running buffer at pH 7.4. Assays were performed in the following steps at 30 °C: (1) equilibration with water for 60 s; (2) activation of AR2G biosensors by 20 mM 1-ethyl- 3-(3-dimethylaminopropyl) carbodiimide hydrochloride and 10 mM N-hydroxysuccinimide for 300s; (3) immobilization of protein onto sensors for 300 s; (4) quenching with 1Methanolamine (pH 8.5) for 300 s; (5) baseline step in 1× Kinetics buffer for 120 s; (6) association of rEF1a for measurement of K_on for 600 s; (7) dissociation of rEF1a for measurement of K_off (700 s). Association and dissociation were measured and fitted based on a 1:1 binding kinetic model with ForteBio Data Analysis software.

Zhou H., Guan Y., Feng M., Fu Y., Tachibana H, & Cheng X. (2020). Evaluation on Elongation Factor 1 Alpha of Entamoeba histolytica Interaction with the Intermediate Subunit of the Gal/GalNAc Lectin and Actin in Phagocytosis. Pathogens, 9(9), 702.

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