Proteon xpr36 instrument

Manufactured by Bio-Rad

Sourced in Israel, United States, Switzerland

The ProteOn XPR36 instrument is a label-free, surface plasmon resonance (SPR) system designed for real-time, quantitative analysis of biomolecular interactions. The instrument uses a proprietary flow cell and detector configuration to enable parallel, multi-channel detection of up to 36 sensor spots. The ProteOn XPR36 is capable of measuring kinetic parameters, affinity, and concentration of a variety of biomolecular interactions, including protein-protein, protein-small molecule, and protein-nucleic acid interactions.

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Lab products found in correlation

Proteon sensor chip, by Bio-Rad (3 mentions) Biacore 3000, by GE Healthcare (2 mentions) Neutravidin immobilized nlc proteon sensor chip, by Bio-Rad (2 mentions) Glm sensor chip, by Bio-Rad (2 mentions) Biacore 3000 instrument, by GE Healthcare (1 mentions) Cm5 sensor chip, by GE Healthcare (1 mentions) Glc sensor chip, by Bio-Rad (1 mentions) Pbs buffer, by Bio-Rad (1 mentions) Biaevaluation version 3, by GE Healthcare (1 mentions) Proteon manager, by Bio-Rad (1 mentions) Prism version, by GraphPad (1 mentions) Human serum albumin (hsa), by Merck Group (1 mentions) Proteon glc sensor chip, by Bio-Rad (1 mentions) Proteon manager v 3, by Bio-Rad (1 mentions) Proteon manager software, by Bio-Rad (1 mentions) Biaevaluation 4, by Cytiva (1 mentions) Empower pro software, by Waters Corporation (1 mentions)

23 protocols using proteon xpr36 instrument

Affibody Binding Kinetics for VEGFR2

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Human VEGFR2/Fc was immobilized on a CM-5 sensor chip (GE Healthcare). Binding kinetics were analyzed by injecting different concentrations of the Affibody molecules (5, 10 and 20 nM) using a BIAcore™ 3000 instrument (GE Healthcare). Binding kinetics for Z_{VEGFR2_40} binding to murine VEGFR2 were determined using a BIAcore™ 3000 instrument (GE Healthcare) as described for human VEGFR2. For determination of the affinity constants of Z_{VEGFR2_22}, Z_{VEGFR2_19} and Z_{VEGFR2_16} for murine VEGFR2, the Affibody molecules were immobilized on a GLC sensor chip (Biorad Laboratories) using a ProteOn XPR36 instrument (Biorad Laboratories). 5, 10 and 20 nM murine VEGFR2 (Sino Biological) was injected over the surfaces. PBST 0.1 was used as a running buffer and 10 mM HCl for regeneration. The experiment was performed in duplicates using freshly prepared reagents.

Fleetwood F., Klint S., Hanze M., Gunneriusson E., Frejd F.Y., Ståhl S, & Löfblom J. (2014). Simultaneous targeting of two ligand-binding sites on VEGFR2 using biparatopic Affibody molecules results in dramatically improved affinity. Scientific Reports, 4, 7518.

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Kinetics of NZM-specific Monoclonal ADAs

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To study the kinetics of interaction of NZM-specific monoclonal ADAs,
NZM IgG (50 nM) was stabilized in 10 mM acetate buffer, pH 4.5, and immobilized
onto a EDC/NHS pre-activated ProteOn sensor chip (Biorad) through amine
coupling; unreacted groups were blocked by injection of ethanolamine HCl (1 M).
HEPES buffered saline (HBS) (10 mM HEPES, pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.005%
surfactant Tween-20) was used as running buffer. All injections were made at
flow rate of 100 μl/min. Monoclonal ADAs were diluted to 30 nM and
injected onto the NZM coated chip; one channel of the chip was injected with HBS
and used as reference for the analysis. Injection time and dissociation time
were 240 s and 600 s, respectively. The binding interaction of each antibodies
with NZM was assessed using a ProteON XPR36 instrument (BioRad). SPR data were
processed with ProteOn Manager Software and k_a (1/Ms), k_d(1/s) and KD (M) parameters were calculated applying the Langmuir fit model.

Cassotta A., Mikol V., Bertrand T., Pouzieux S., Le Parc J., Ferrari P., Dumas J., Auer M., Deisenhammer F., Gastaldi M., Franciotta D., Silacci-Fregni C., Fernandez Rodriguez B., Giacchetto-Sasselli I., Foglierini M., Jarrossay D., Geiger R., Sallusto F., Lanzavecchia A, & Piccoli L. (2019). A single T cell epitope drives the neutralizing anti-drug antibody response to natalizumab in multiple sclerosis patients. Nature medicine, 25(9), 1402-1407.

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Affibody-HPV16 E7 Protein Binding Kinetics

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A ProteOn XPR36 instrument (Bio-Rad) was used for real-time biospecific interaction analysis between selected affibody molecules and the target protein. HPV16 E7 protein diluted in 10 mmol/L NaAc (pH 4.5) was immobilized into the surface of carboxylate glucans in HTG sensor chip according to the manufacturer's instructions. Another flow-cell surface was activated and deactivated to be used as a reference controls. Different concentration of Z_{HPV16 E7}127, Z_{HPV16 E7}301, Z_{HPV16 E7}384 and Z_{HPV16 E7}745, ranging from 1.0 nM to 64.0 nM, were injected over all surfaces with a flow rate of 30 μL/min. Wild SPA-Z (Zwt) affibody was set as negative control. Binding analyses were done at 25°C, and PBS was used as the running buffer. The dissociation equilibrium constant (KD), the association rate constant (ka), and the dissociation rate constant (kd) were calculated using BIA evaluation 3.0.2 software (Biacore). A one-to-one Langmuir binding model was used to assume the mass transfer effects into account.

Xue X., Wang B., Du W., Zhang C., Song Y., Cai Y., Cen D., Wang L., Xiong Y., Jiang P., Zhu S., Zhao K.N, & Zhang L. (2016). Generation of affibody molecules specific for HPV16 E7 recognition. Oncotarget, 7(45), 73995-74005.

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Investigating Helicase-DNA Interactions via SPR

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Surface plasmon resonance (SPR) was measured using a Proteon XPR36 instrument (Bio-Rad). The measurements were performed at 30°C in a PBS buffer (Bio-Rad) added with 0.01% of Tween 20 (PBST) and with or without ATP 0.5 mM. NLC sensor chips (Bio-Rad) were used to immobilize the oligonucleotides oso13 or oso15 (Supplementary Table S1) through their biotin-tag. For immobilization, DNA were diluted in PBST and attached to the chip to obtain 50 RU in different orientations (3′ free for oso13 or 5′ free for oso15). For the observation of kinetic data, two different kinds of experiments were performed. In experiments where loading and translocation were coupled, proteins were injected in PBST with 0.5 mM ATP and 10 mM MgCl₂ at 50 μl/min during 240 sec, and dissociation was run during 900 sec. In experiments where loading and translocation of the helicase on DNA were uncoupled, proteins were injected in PBST with 0.5 mM ATP during same duration, and the 900 sec dissociation was followed by injection of PBST with ATP 0.5 mM plus MgCl₂ 10 mM during 459 sec. In all experiments, the proteins were injected at different concentrations and different molar ratios, as indicated in the Results section. After each interaction test, the chip was regenerated using 0.5% of SDS. After corrected by subtraction of the uncoated reference channel, the sensorgrams were analyzed and compared.

Marsin S., Adam Y., Cargemel C., Andreani J., Baconnais S., Legrand P., Li de la Sierra-Gallay I., Humbert A., Aumont-Nicaise M., Velours C., Ochsenbein F., Durand D., Le Cam E., Walbott H., Possoz C., Quevillon-Cheruel S, & Ferat J.L. (2021). Study of the DnaB:DciA interplay reveals insights into the primary mode of loading of the bacterial replicative helicase. Nucleic Acids Research, 49(11), 6569-6586.

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Quantifying EGFR and EGFRvIII Binding Kinetics

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The experiment was performed using ProteOn XPR36 instrument (Bio‐Rad, Hercules) at 25°C in phosphate‐buffered saline (PBS) containing 0.05% Tween (PBS‐T). Streptavidin was diluted into 10 mm sodium acetate (pH 4.0), and 300 response units (RU) were immobilised to three flow cells of the GLC sensor chip by amine coupling. 200–400 RU of biotinylated GCT02 and GFP scFvs were captured onto separate flow cells of the streptavidin‐coupled sensor chip. A separate flow cell containing immobilised streptavidin alone served as a control channel.
Recombinant EGFR and EGFRvIII (ACROBiosystems, Newark HEK293T source, HIS tag) protein was reconstituted at 1 μg μL⁻¹ in PBS before dilution into PBS with 0.05% Tween and injection over the sensor chip at the concentrations indicated (Figure 1a) (flow rate 30 μL per min). GFP protein controlled for non‐specific scFv binding. 10 mm glycine HCl buffer (pH 3) was used to strip injected protein from the chip between injections. Results from at least two independent injections were analysed. After subtraction of data from control cells, interactions were analysed with ProteOn Manager software (version 2.1). The equilibrium dissociation constant (KD) values were derived from kinetic fit analysis using 1:1 Langmuir binding model.

Abbott R.C., Verdon D.J., Gracey F.M., Hughes‐Parry H.E., Iliopoulos M., Watson K.A., Mulazzani M., Luong K., D’Arcy C., Sullivan L.C., Kiefel B.R., Cross R.S, & Jenkins M.R. (2021). Novel high‐affinity EGFRvIII‐specific chimeric antigen receptor T cells effectively eliminate human glioblastoma. Clinical & Translational Immunology, 10(5), e1283.

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SPR-based Protein-Protein Interaction Assays

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SPR-based measurements
of protein–protein interactions were performed on a Biacore
3000 (GE Healthcare) and a Bio-Rad ProteOn XPR36 instrument, essentially
as described previously.^{20 (link)} Briefly, purified
His₆-EhPAK4 PBD and His₆-EhPAK5 PBD proteins
were separately immobilized on an NTA biosensor chip using covalent
capture coupling.^{31 (link)} EhRacC, EhRacC^Q65L, EhRacD, EhRacG, or EhRho1 was injected in 30–100
μL volumes at increasing concentrations. Experiments were performed
in a running buffer containing 50 mM HEPES (pH 7.4), 150 mM NaCl,
0.05% NP-40 alternative (Calbiochem), 50 μM EDTA, and 1 mM MgCl₂. Background changes in refractive index upon injection of
samples were subtracted from all curves using BIAevaluation version
3.0 (GE Healthcare) or ProteOn Manager (Bio-Rad). Equilibrium binding
analyses were conducted as previously described^{32 (link)} using GraphPad Prism version 5.0 to determine binding affinities.
Kinetic analyses were performed on triplicate Rho GTPase injections
as previously described.^{33 (link)}

Bosch D.E, & Siderovski D.P. (2014). Entamoeba histolytica RacC Selectively Engages p21-Activated Kinase Effectors. Biochemistry, 54(2), 404-412.

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SPR Analysis of VEGFR2 Binding

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The binding of the four dimeric constructs to human and murine VEGFR2 was analyzed by SPR. HSA (Sigma-Aldrich) was immobilized on a GLM sensor chip (Biorad Laboratories) using a ProteOn XPR36 instrument (Biorad Laboratories). The apparent dissociation constants (k_d) were estimated by injections of 200 nM of each of the homodimeric and heterodimeric constructs over the immobilized HSA, immediately followed by injections of 5, 15 and 45 nM human or murine VEGFR2 (for the heterodimers), or 15, 30 and 60 nM of human or murine VEGFR2 (for the homodimers). The dissociation constants were obtained from sensorgrams using a monovalent binding equation and non-linear regression. The experiment was performed in duplicates using freshly prepared reagents.

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Kinetic Characterization of Anti-GM-CSF Antibodies

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Protein A (450 nM) was stabilized in 10 mM acetate buffer, pH 4.5, and immobilized onto a EDC/NHS pre-activated ProteOn sensor chip (Biorad) through amine coupling; unreacted groups were blocked by injection of ethanolamine HCl (1 M). HEPES buffered saline (HBS) (10 mM HEPES, pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.005% surfactant Tween-20) was used as running buffer. All injections were made at flow rate of 100 μl min⁻¹. Monoclonal antibodies were diluted in HBS (200 nM) and injected onto the protein A coated chip for capturing, followed by injection of different concentrations of human GM-CSF (400 nM, 200 nM, 100 nM, 50 nM, 25 nM); one channel of the chip was injected with HBS and used as reference for the analysis. Injection time and dissociation time were 120 s and 600 s, respectively. Each binding interaction of mAbs with GM-CSF was assessed using a ProteON XPR36 instrument (Biorad) and data processed with ProteOn Manager Software. Ka, Kd and KD were calculated applying the Langmuir fit model. To determine the epitope specificity, GM-CSF autoantibodies (150 mM) were directly immobilized on a sensor chip, followed by injection of GM-CSF (100 nM) and autoantibodies (200 nM). To assess simultaneous binding to GM-CSF, different mAbs (200 nM each) were serially injected after GM-CSF capture (50 nM). Injection time and dissociation time were 60 s and 20 s, respectively.

Piccoli L., Campo I., Fregni C.S., Rodriguez B.M., Minola A., Sallusto F., Luisetti M., Corti D, & Lanzavecchia A. (2015). Neutralization and clearance of GM-CSF by autoantibodies in pulmonary alveolar proteinosis. Nature Communications, 6, 7375.

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Kinetic Analysis of Monoclonal Antibodies

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Biotinylated NPDP15 and NANP18 peptides were diluted (20 nM) in HEPES buffered saline (HBS) (10 mM HEPES, pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.005% surfactant Tween-20). HBS was also used as running buffer. An irrelevant biotinylated 19-mer peptide was used as a control for non-specific interactions. A NeutrAvidin-immobilized NLC ProteOn sensor chip (Biorad) was pre-conditioned with an NaCl solution (1 M) and the biotinylated peptides were injected onto the chip. The monoclonal antibodies were diluted and titrated in HBS (50-16.7-5.6-1.9-0.6 nM) and injected onto chip; one channel of the chip was injected with HBS and used as reference for the analysis. All injections were made at a flow rate of 100 μl/min. Injection time and dissociation time were 240 s and 900 s, respectively. Each binding interaction of the monoclonal antibodies with the biotinylated peptides was assessed using a ProteON XPR36 instrument (Biorad) and data were processed with ProteOn Manager Software (version 3.1.0.6). K_a, K_d and K_D were calculated by applying the Langmuir fit model.

Tan J., Sack B.K., Oyen D., Zenklusen I., Piccoli L., Barbieri S., Foglierini M., Fregni C.S., Marcandalli J., Jongo S., Abdulla S., Perez L., Corradin G., Varani L., Sallusto F., Sim B.K., Hoffman S.L., Kappe S.H., Daubenberger C., Wilson I.A, & Lanzavecchia A. (2018). A public antibody lineage that potently inhibits malaria infection by dual binding to the circumsporozoite protein. Nature medicine, 24(4), 401-407.

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Surface Plasmon Resonance Analysis of Fluorescent Proteins

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Surface plasmon resonance was measured using a Proteon™ XPR36 instrument (Bio-Rad). All measurements were performed in 50 mM phosphate buffer, pH 7, 150 mM NaCl and 0.005% Tween 20 at a flow rate of 100 μl·min⁻¹. ProteOn GLC sensor chip (Bio-Rad) were used to immobilized αRep proteins (bGFP-A, bGFP-C, bGFP-D and αRep A3) in parallel on one of the six channels chip following the amine-coupling protocol. For the determination of kinetics data, purified FPs (EGFP, ECFP, EYFP and mCherry) were injected each at six different concentrations in parallel (0; 1.1; 3.3; 10; 30 and 90 nM) during 200 s and dissociation signals were acquired during 600 s. The signal of the uncoated reference channel and interspots were always subtracted from the sensorgrams. The kinetic data were analysed with the Proteon Manager software fitted by Langmuir analysis for the five protein concentrations.

Chevrel A., Urvoas A., de la Sierra-Gallay I.L., Aumont-Nicaise M., Moutel S., Desmadril M., Perez F., Gautreau A., van Tilbeurgh H., Minard P, & Valerio-Lepiniec M. (2015). Specific GFP-binding artificial proteins (αRep): a new tool for in vitro to live cell applications. Bioscience Reports, 35(4), e00223.

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